1. Developmental Biology
  2. Immunology and Inflammation
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Diverse homeostatic and immunomodulatory roles of immune cells in the developing mouse lung at single cell resolution

  1. Racquel Domingo-Gonzalez
  2. Fabio Zanini
  3. Xibing Che
  4. Min Liu
  5. Robert C Jones
  6. Michael A Swift
  7. Stephen R Quake  Is a corresponding author
  8. David N Cornfield  Is a corresponding author
  9. Cristina M Alvira  Is a corresponding author
  1. Division of Critical Care Medicine, Department of Pediatrics, Stanford University School of Medicine, United States
  2. Center for Excellence in Pulmonary Biology, Stanford University School of Medicine, United States
  3. Department of Bioengineering, Stanford University, United States
  4. Prince of Wales Clinical School, Lowy Cancer Research Centre, University of New South Wales, Australia
  5. Division of Pulmonary, Asthma and Sleep Medicine, Department of Pediatrics, Stanford University School of Medicine, United States
  6. Department of Chemical and Systems Biology, Stanford University, United States
  7. Chan Zuckerberg Biohub, United States
  8. Department of Applied Physics, Stanford University, United States
Research Article
Cite this article as: eLife 2020;9:e56890 doi: 10.7554/eLife.56890
9 figures, 12 tables, 3 data sets and 1 additional file

Figures

Figure 1 with 2 supplements
Diversity of the lung immune landscape increases dramatically after birth.

(A) Overview of the experimental design including the four timepoints (E18.5, P1, P7, P21) corresponding to key stages in late lung development. Lungs were isolated, perfused, and digested and immune cells isolated by fluorescence activated cell sorting (FACS) for the dead-stain-, EPCAM-, CD45+ population. (B) t-Distributed Stochastic Neighbor Embedding (t-SNE) and unsupervised clustering of over 4000 immune cells identifies fifteen distinct populations. (C) Dot plot showing level of expression (purple to yellow), and fraction of the population expressing the particular gene (dot size) for distinguishing genes expressed by the Leiden clusters broadly separated into myeloid (M), dendritic cell (DC) granulocyte (G) and lymphocyte (L) populations. (D) t-SNE of immune cell clusters identifying developmental timepoint of cell origin with E18.5 (blue), P1 (yellow), P7 (orange) and P21 (red). (E) Quantification of the abundance of specific immune subpopulations in the lung at each developmental timepoint expressed on a log (Rodríguez-Castillo et al., 2018) scale as percentage of total immune cells.

Figure 1—figure supplement 1
Optimization of lung tissue digestion.

(A–C) E18.5, P1, P7 and P21 murine lungs were processed for flow cytometry and the frequency of (B) total and (C) CD45+ (immune) dead cells was assessed. (D–E) Frequency of dead cells of P7 murine lungs was determined by flow cytometry following 30 min (D) enzymatic digestion with 0.38 mg/mL liberase with manual tituration (Liberase) and/or mechanical disruption using the lung program on the GentleMACS dissociator (Lung GM) or (E) enzymatic digestion with collagenase and manual tituration (Collagenase), Liberase, or 0.38 mg/mL liberase with the Multi_D program on the GentleMACS dissociator (Liberase + Multi_D GM). (F) Number of viable P7 murine lung cells following Collagenase, Liberase, or Liberase + Multi_D GM was quantified. (G) Frequency of murine lung cells at gestational age E18.5, P1, P21 was quantified following incubation with 0.38 mg/mL liberase for 15, 25, and 30 min and manual tituration.

Figure 1—figure supplement 2
Determination of variation between mice.

To quantify whether the different mice contributed spurious variation to the data, a distribution level approach was chosen. For each cell type and time point, 100 pairs of cells from either the same mouse or between different mice were chosen and the distance in t-SNE space calculated. The cumulative distributions for those pairs were subsequently plotted to check whether pairs from different animals had a significantly longer distance than cells from the same mouse.

Figure 2 with 2 supplements
Macrophage populations present before and after birth broadly separate into two populations based on expression of Dab2 and Plac8.

(A) t-SNE plot depicting Cd68 expression in the five macrophage populations. (B) Separate embeddings for prenatal versus postnatal macrophages, identifying developmental timepoint of cell origin with E18.5 (blue), P1 (yellow), P7 (orange) and P21 (red). (C) Quantification of the abundance of each macrophage subpopulation at each developmental timepoint expressed on a log (Rodríguez-Castillo et al., 2018) scale as percentage of total macrophages. (D) t-SNE plots depicting expression of Dab2 and Plac8 within the macrophages present pre- and post-birth. (E) Multiplexed in situ hybridization to detect gene expression of Cd68 (yellow), Dab2 (red), and Plac8 (green) in lung tissue from mice at E18.5 and P7. Quantification of Dab2+ and Plac8+ cells with data = mean ± SD for n = 4–15 images from three separate FISH experiments. Calibration bar = 20 μm. For all t-SNE embeddings, the color scale is a logarithmic scale with a pseudocount of 0.1 counts per million, normalized to the highest expressing cell. Source files of all fluorescent micrographs used for the quantitative analysis are available in the Figure 2—source data 1.

Figure 2—source data 1

Source files for quantification of Dab2+ and Plac8+ Cd68+ cells.

This zip archive contains all the fluorescent micrographs used for the quantitative analysis shown in Fig. blank. The individual files are named with the timepoint (for figures containing more than one timepoint), the gene detected by FISH, followed by the color of the label for the gene with ‘G’ for green, ‘R’ for red, ‘W’ for white, and ‘Y’ for yellow.

https://cdn.elifesciences.org/articles/56890/elife-56890-fig2-data1-v2.zip
Figure 2—figure supplement 1
Multiplex in situ hybridization to detect Dab2 and Plac8 expressing macrophages.

In situ hybridization at E18.5 and P7 to detect Cd68 (white), Dab2 (red), Plac8 (green), and a merged image. Calibration bar = 20 μm.

Figure 2—figure supplement 2
Lineage-defining genes are diffusely expressed across macrophage populations.

t-SNE plots of Adgre1, Lgals3, Mertk, Ly6c2, Ly6c1 and Itgam expression across all macrophage clusters between E18.5 and P21. The color scale is a logarithmic scale with a pseudocount of 0.1 counts per million, normalized to the highest expressing cell.

Figure 3 with 1 supplement
Embryonic macrophages encircle developing blood vessels prior to birth.

(A) t-SNE plots depicting expression of Mki67 and Mcm5 in the macrophage clusters with low expression in purple and high expression in yellow, with quantification of proliferating macrophages at each timepoint. In situ hybridization at E18.5 to detect: (B) Cd68 (red) and Cdh5 (green) with dotted lines identifying macrophages in circles around small vessels; (C) Epcam (green), Cd68 (red), and extracellular matrix (ECM, yellow), with white dotted circles identifying Cd68+ cells or groups of cells; (D) Quantification of the number of Cd68+ cells around vessels versus in lung parenchyma, with data = mean ± SD in n = 14 images, from five independent FISH experiments. (E) In situ hybridization at E18.5 to detect Mki67 (red), Cd68 (green), and ECM (yellow), with white dotted circles identifying Cd68+Mki67+ cells, and solid circles identify Cd68+ Mki67- cells. Quantification of the number of perivascular and parenchymal Mki67+ and Mki67- Cd68+ cells in n = 10 images with data = mean ± SD from two independent FISH experiments. (F) In situ hybridization at E18.5 to detect Cd68 (white) Dab2 (red), and Plac8 (green) with white dotted circles identifying Cd68+Dab2+ macrophages in the left panel, and arrows identifying Plac8+ cells in a separate area from the same slide (right panel). (G) In situ hybridization at E18.5 to detect Cd68 (red), Gal (white), and C1qa (green), with quantification of Gal+ and C1qa+ Cd68+ cells with data = mean ± SD from n = 12 images from three independent FISH experiments. (H) t-SNE plots of genes previously associated with a perivascular macrophage phenotype. (I) In situ hybridization of lung at P1 to detect Cd68 (red) and ECM (yellow), with white dotted circles identifying isolated macrophages around blood vessels. In each micrograph, calibration bar = 20 μm and ‘V’ denotes ‘vessel and ‘A’ ‘airway’. For all t-SNE embeddings, the color scale is a logarithmic scale with a pseudocount of 0.1 counts per million, normalized to the highest expressing cell. Source files of all fluorescent micrographs used for the quantitative analysis are available in the Figure 3—source data 1, Figure 3—source data 2, and Figure 3—source data 3.

Figure 3—source data 1

Source files for quantification of perivascular and parenchymal Cd68+ cells at E18.5.

This zip archive contains all the fluorescent micrographs used for the quantitative analysis shown in Fig. blank. The individual files are named with the timepoint (for figures containing more than one timepoint), the gene detected by FISH, followed by the color of the label for the gene with ‘G’ for green, ‘R’ for red, ‘W’ for white, and ‘Y’ for yellow.

https://cdn.elifesciences.org/articles/56890/elife-56890-fig3-data1-v2.zip
Figure 3—source data 2

Source files for quantification of Mki67+ Cd68+ cells at E18.5.

This zip archive contains all the fluorescent micrographs used for the quantitative analysis shown in Fig. blank. The individual files are named with the timepoint (for figures containing more than one timepoint), the gene detected by FISH, followed by the color of the label for the gene with ‘G’ for green, ‘R’ for red, ‘W’ for white, and ‘Y’ for yellow.

https://cdn.elifesciences.org/articles/56890/elife-56890-fig3-data2-v2.zip
Figure 3—source data 3

Source files for quantification of Gal+ and C1qa+ perivascular Cd68+ cells at E18.5.

This zip archive contains all the fluorescent micrographs used for the quantitative analysis shown in Fig. blank. The individual files are named with the timepoint (for figures containing more than one timepoint), the gene detected by FISH, followed by the color of the label for the gene with ‘G’ for green, ‘R’ for red, ‘W’ for white, and ‘Y’ for yellow.

https://cdn.elifesciences.org/articles/56890/elife-56890-fig3-data3-v2.zip
Figure 3—figure supplement 1
Distribution of proliferating cells among macrophage/monocyte clusters.

Graph of percentage of proliferating macrophages in each cluster at E18.5.

Figure 4 with 4 supplements
Distinct transcriptional profiles and spatial distribution suggest specific physiologic functions for discrete macrophage populations.

(A) Heatmap of select differentially expressed genes within enriched pathways illustrated. The color scale is logarithmic with a pseudocount of 0.1 counts per million, normalized to the highest expressing population for each gene, depicting 0 (none), 33 (low), 67 (mid), and 100% (high) expression compared to the highest expressing population. (B) t-SNE plots demonstrating high expression of Gal in Mac I cells, and in situ hybridization at E18.5 to detect Mac I cells that co-express Gal (red) and Cd68 (green). (C) t-SNE plots demonstrating high expression of Itgax but not Car4 in Mac II cells, and in situ hybridization to detect Mac II cells at P1 expressing Itgax, and Cd68 but not Car4 (dotted line), and additional Mac III cells in the same image co-expressing Itgax, Cd68, and Car4 (arrow). (D) t-SNE plots demonstrating high expression of Itgax and Car4 in Mac III cells, and in situ hybridization detecting Mac III cells at P7 expressing Itgax, Car4 and Cd68 now located within alveoli. (E) t-SNE plot demonstrating high expression of C1qa in Mac IV cells, and in situ hybridization to detect Mac IV cells expressing C1qa (green) and Cd68(red) at P7, with ECM marked in yellow, localizing Mac IV cells abutting vessels and large airways. Calibration bar = 20 μm for all panels. For all t-SNE embeddings, the color scale is a logarithmic scale with a pseudocount of 0.1 counts per million, normalized to the highest expressing cell. (F) t-SNE plot showing a developmental gradient across Mac I-III. Pseudotime ordering of the cells identified a global trajectory from the starting cell (star) in Mac I to Mac III, and local cell states revealing multiple areas of local attraction within the Mac II and Mac III clusters. (G) RNA velocity demonstrating local group velocity from Mac I to Mac II and continuation of the vectors through Mac III.

Figure 4—figure supplement 1
Pathway analysis of differentially expressed genes in the macrophage populations.

Pathway analysis on the top 100 expressed genes from the 5 Mac clusters was performed for each cluster in a one-vs-rest comparison, and the gene set enrichment evaluated with Metascape. Enriched pathways were ordered by significance. The x axis is the negative logarithm of the P value.

Figure 4—figure supplement 2
Select genes involved in pathways regulating lung progenitor cells are expressed by Mac II and Mac IV.

(A) t-SNE plots of genes expressed by Mac II including Ntn1, Ntn4, Hhip, Tgfb1, and Tgfb2 with plot of cell subtype. (B) t-SNE plots of genes expressed by Mac IV including Fgf10, Bmp4, Wnt2, and WNt5a. For all t-SNE plots, the color scale is a logarithmic scale with a pseudocount of 0.1 counts per million, normalized to the highest expressing cell, after smoothing with a modified version of the MAGIC algorithm as described in the Materials and methods.

Figure 4—figure supplement 3
Expression of select genes in Mac IV cluster.

t-SNE plots of (A) H2-Ab1, (B) H2-Eb1, (C) Cd74, and (D) Mrcl1 demonstrating differential expression in a portion of Mac IV (dotted red line). t-SNE plots of (E) Cx3cr1, (F) Mertk, (G) Cclr2, and (H) Cd68 demonstrating diffuse expression throughout Mac IV. The color scale is a logarithmic scale with a pseudocount of 0.1 counts per million, normalized to the highest expressing cell.

Figure 4—figure supplement 4
Multiplex in situ hybridization to detect specific macrophage clusters.

(A) In situ hybridization at P1 to detect Cd68 (red), Itgax (white), Car4 (green) and a merged image. (b) In situ hybridization at P1 to detect Cd68 (white), Ifitm6 (red), Ccr2 (green) and a merged image. In all images, calibration bar = 20 μm.

The Mac V cluster harbors distinct subpopulations with developmentally regulated gene expression patterns.

(A) t-SNE plots demonstrating high expression of Plac8 and Ifitm6 in Mac V cells. (B) t-SNE plots of Ccr1, Ccr2, Ace, and Slc12a2 suggesting the presence of two transcriptionally distinct populations within the Mac V cluster. (C) t-SNE demonstrating a developmental gradient within the Mac V sub cluster. For all t-SNE embeddings, the color scale is a logarithmic scale with a pseudocount of 0.1 counts per million, normalized to the highest expressing cell. (D) Pseudotime analysis with the star indicating the starting point and the arrow denoting the trajectory across pseudotime. (E) Heatmap of differentially expressed genes within enriched pathways across pseudotime. The color scale is logarithmic with a pseudocount of 0.1 counts per million, normalized to the highest expressing population for each gene, depicting 0 (none), 33 (low), 67 (mid), and 100% (high) expression compared to the highest expressing population. (F) In situ hybridization of Cd68 (white), Ifitm6 (red), and Ccr2 (green), and to detect the ‘early’ Mac V subcluster at P1. (G) In situ hybridization of Cd68 (white), Lair1 (red), and Ifitm6 (green), to detect the ‘late’ Mac V subcluster at P21. Calibration bar = 20 μm for all panels.

Comparison of Macrophage/Monocyte Subtypes in the Perinatal and Adult Lung.

Data sets were merged, restricted to genes that were overlapping, normalized to counts per million reads, feature selected and subjected to PCA, followed by UMAP. UMAP plots of (A) cell type and (B) data source using our data combined with Schyns et al. Data sets were merged and processed as described above, and the Northstar algorithm was used to infer the cell subtypes for our cells based on the Tabula Muris Atlas, with UMAP plots of (C) cell type and (D) data source.

Expression of Fc receptors across macrophage clusters.

t-SNE plots of the expression of Fc receptors Fcgr1, Fcgr2b, Fcgr3, Fcgr4, Fcer1a, Fcer1g, Fcer2a, Fcgrt. For all tSNE embeddings, the color scale is a logarithmic scale with a pseudocount of 0.1 counts per million, normalized to the highest expressing cell.

Figure 8 with 1 supplement
Multiple dendritic cell populations and lung granulocytes are primed for rapid pathogen response.

(A) Colored schematic and lung immune cell clustering demonstrating three separate clusters of DCs. (B) t-SNE plots of genes discriminating the three DC subclusters including Itgae (cDC1), Cd209a (cDC2) and Mreg (mig-DC). (C) Quantification of specific DC subpopulations relative to total immune cells (left) or total DC (right). (D) Colored schematic and lung immune cell clustering demonstrating the basophil, mast cell and neutrophil clusters, with high magnification of basophil and mast cell clusters and t-SNE plots of Mcpt4 and Mcpt8. (E) t-SNE plots of Il6, Hgf, Ccl4, and Osm in the basophil cluster. (F) High magnification of the neutrophil cluster with t-SNE plots of neutrophil specific genes S100a8, S100a9, Stfa1, and Stfa2. For all t-SNE embeddings, the color scale is a logarithmic scale with a pseudocount of 0.1 counts per million, normalized to the highest expressing cell.

Figure 8—figure supplement 1
Expression of dendritic cell associated genes.

t-SNE plots of (A) pan-DC-associated genes Zbtb46 and Flt3; (B) cDC1 specific genes Itgae, Irf8, Xcr1, and Cadm1; (C) cDC2 specific genes Itgam, Sirpa, and Irf4; (D) monocyte-derived specific genes Fcer1g, Fcgr1, Ly6c1, and Ly6c2; and (E) mig-DC associated genes Cacnb3, Fscn1, Ccl5, and Ccr7.

Figure 9 with 1 supplement
Lymphocytes populate the lung at birth but remain naïve during the first three weeks of life (A) Quantification of the abundance of B and T cells at each developmental timepoint expressed on a log (Rodríguez-Castillo et al., 2018) scale as percentage of total immune cells.

(B) Quantification of the percentage of proliferating B cells at each developmental timepoint. (C) B cell heavy and light variable (V) and joining (J) gene identity and their cumulative cell fraction. (D) t-SNE plot of Ighm and Ighd, with the logarithmic color scale with a pseudocount of 0.1 counts per million, normalized to the highest expressing cell. (E–H) At P1 (n = 7 mice) and P21 (n = 8 mice), lungs were processed to a single-cell suspension, and flow cytometry was used to assess frequencies of (F) CD4+CD3+ and CD8+CD3+ T cells, (G) Gata3+, Tbet+, Rorγt+ and Foxp3+ CD4+ T cells, and (H) IL-4-producing Gata3+CD4+ T cells or IFNγ-producing Tbet+CD4+ T cells. Data shown as mean ± SD, **p<0.01, ****p<0.00001 by Student’s t test.

Figure 9—figure supplement 1
Transcriptional and flow cytometric profiling of lymphocytes.

t-SNE plots of (A) B cell-associated genes Aicda, Tbx21, Prdm1, and Ebi3. (B) Clonality and t-SNE plots of Mki67 and Syk within the B cell cluster. (C) t-SNE plots of T cell-associated genes Trac and Tcrg-C4. (D) Bar graph of the frequencies of Cd4+Cd8-, Cd4-Cd8+, Cd4-CD8-, and Cd4+Cd8+ T cells at P1, P7, and P21. (E-F) Flow cytometric analyses of murine lung cells isolated at P1 and P21 assessing the frequencies of (E) CD3+ lymphocytes and (F) Il-17-producing Th17 cells and IL-10-producing T regulatory cells.

Tables

Table 1
Top 35 differentially expressed genes in the Mac I cluster.

ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 35 upregulated genes are shown for the Mac I cluser; n = 2 mice.

Mac I cluster top 35 differentially expressed genes
Gene nameStatisticP valueLog2 fold change
Gal0.61335.42E-533.6207
Crispld20.51.89E-343.6018
Ncapd20.45671.40E-283.3349
Pclaf0.494.88E-333.1388
Mcm50.50672.07E-352.9792
Cdca80.441.74E-262.8548
Lig10.459.89E-282.7655
AI5068160.52337.05E-382.7046
Asf1b0.44336.73E-272.5712
Smc20.43674.44E-262.4042
Fkbp50.55331.42E-422.3039
Mcm70.441.74E-262.1166
Cks1b0.459.89E-282.0842
A930007I19Rik0.465.23E-292.0187
Psat10.522.24E-372.0158
Pfkl0.44672.59E-271.9417
Ezh20.44336.73E-271.8453
Dkc10.47339.21E-311.8027
Cluh0.441.74E-261.7959
Scd20.441.74E-261.7455
Ruvbl20.44336.73E-271.7334
Mif0.49331.67E-331.6941
Tmem2730.441.74E-261.628
Stmn10.44336.73E-271.5905
Spint10.52672.20E-381.5781
Tpi10.50336.29E-351.562
C230062I16Rik0.459.89E-281.5462
Nt5dc20.44672.59E-271.5376
Ran0.45671.40E-281.5078
Trf0.59332.32E-491.4397
Gcnt10.441.74E-261.4095
Abcd20.465.23E-291.3537
Anp32b0.472.56E-301.3418
Dbi0.51677.04E-371.2018
Atp5g30.522.24E-371.1808
Table 2
Top 35 differentially expressed genes in the Mac II cluster.

ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 35 upregulated genes are shown for the Mac II cluster; n = 2 mice.

Mac II cluster top 35 differentially expressed genes
Gene nameStatisticP valueLog2 fold change
Adgrl30.57138.85E-423.8593
Adarb10.42165.92E-232.6952
Ms4a8a0.45282.11E-262.4485
Nav20.47514.97E-292.1319
Tmcc30.42899.85E-242.0579
Ldhb0.38098.26E-192.0235
Acp50.38374.45E-192.0198
Dapk10.36582.20E-171.8704
Itgax0.58252.02E-431.8680
Csf2rb20.34591.11E-161.7791
H2-DMa0.50222.22E-321.7642
Trerf10.38503.31E-191.7402
Dmxl20.41176.59E-221.7264
Tgm20.36631.99E-171.6906
Ear20.51593.79E-341.6602
Dok20.36951.00E-171.6373
Flt10.36552.34E-171.5457
Axl0.50685.74E-331.4698
AU0202060.37235.40E-181.4207
Ece10.39622.54E-201.4075
Ear100.39384.41E-201.4069
Il180.40841.46E-211.4021
Clec7a0.49591.37E-311.3413
Spint10.37245.29E-181.3407
Mpeg10.54544.05E-381.3269
Il1rn0.34631.11E-161.2922
Clec4n0.44541.45E-251.2765
Fn10.40881.34E-211.2497
Il1b0.40021.02E-201.2281
Dst0.44846.72E-261.1994
F11r0.41294.97E-221.1982
Nceh10.35482.24E-161.1299
Neurl30.37215.74E-181.1091
Tnfaip20.37851.40E-181.0880
Plet10.44611.23E-251.0845
Table 3
Top 35 differentially expressed genes in the Mac III cluster.

ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 35 upregulated genes are shown for Mac III cluster; n = 2 mice.

Mac III cluster top 35 differentially expressed genes
Gene nameStatisticP valueLog2 fold change
Slc39a20.66003.98E-629.2983
Atp6v0d20.95331.14E-1517.1784
Krt190.75331.56E-836.8066
Cd200r40.75331.56E-835.9139
Gpnmb0.69675.32E-705.0666
Ear10.62671.66E-554.9379
Car40.75001.10E-824.6965
Ly750.78672.26E-924.4964
Bhlhe410.78001.51E-904.4452
Slc7a20.79333.16E-944.1798
Lrp120.65339.21E-613.7640
Ccl60.87004.58E-1183.7161
Spp10.62671.66E-553.4907
Serpinb1a0.66003.98E-623.4778
Mgll0.80674.96E-983.4554
Il1rn0.65671.93E-613.2640
Kcnn30.76003.03E-853.2372
Ralgds0.70009.49E-713.1251
Dst0.76334.13E-863.1042
Atp10a0.66003.98E-623.0453
Ccnd20.72333.77E-762.9813
Fabp50.62337.18E-552.9667
Cd360.76003.03E-852.9174
Vat10.74677.57E-822.8682
Myof0.76003.03E-852.8483
Plet10.75672.19E-842.7934
Hvcn10.69675.32E-702.7689
Fth-ps30.69332.94E-692.7627
Lima10.65339.21E-612.7359
Itgax0.75672.19E-842.7210
Card110.63671.93E-572.6520
Mir692-20.75672.19E-842.5974
Ctsd0.81675.70E-1012.5402
Klhdc40.65004.35E-602.5367
Mir692-10.65004.35E-602.4553
Table 4
Top 35 differentially expressed genes in the Mac IV cluster.

ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 35 upregulated genes are shown for the Mac IV cluster; n = 2 mice.

Mac IV cluster top 35 differentially expressed genes
Gene nameStatisticP valueLog2 fold change
Fcrls0.73672.28E-7912.2139
Pf40.79002.70E-9311.4117
Gas60.64339.38E-599.6283
C1qc0.87333.18E-1199.1247
Ccl120.64004.28E-588.7381
C1qa0.87004.58E-1188.3676
Stab10.80334.55E-977.6076
C1qb0.86001.17E-1147.0450
Sdc40.72002.32E-756.3860
Igfbp40.74335.15E-816.2878
Ms4a70.72002.32E-756.0618
Slc40a10.60333.72E-516.0358
Tmem176b0.86001.17E-1145.5402
Maf0.75001.10E-825.4450
S1pr10.63671.93E-575.3458
Igf10.67003.28E-644.8862
Tmem370.59009.02E-494.8077
Ophn10.59332.32E-494.6672
Tmem176a0.71671.41E-744.6367
C3ar10.71004.98E-734.5725
Smagp0.67003.28E-644.5052
Cxcl160.58004.98E-474.2439
Sesn10.56333.27E-444.0183
Apoe0.76003.03E-854.0149
Hpgds0.63338.62E-573.9920
Lgmn0.72676.04E-773.9551
Ms4a4a0.56679.10E-453.5567
Selenop0.62003.07E-543.4452
Cx3cr10.60001.49E-503.3845
Abca10.54335.69E-413.2834
Tcf40.54335.69E-413.1404
Blvrb0.55331.42E-423.1103
Aif10.69332.94E-693.0826
Abca90.58331.32E-473.0325
Pea15a0.54001.91E-403.0198
Table 5
Top 35 differentially expressed genes in the Mac V cluster.

ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 35 upregulated genes are shown for the Mac V cluster; n = 2 mice.

Mac V cluster top 35 differentially expressed genes
Gene nameStatisticP valueLog2 fold change
I830127L07Rik0.48671.42E-327.0634
Ifitm60.88001.41E-1216.9362
Pglyrp10.68678.67E-686.2150
Serpinb100.53332.09E-396.1537
Plac80.96006.02E-1555.9647
Adgre40.60333.72E-515.8672
Nxpe40.50001.89E-344.8441
Ifitm30.92671.17E-1394.6727
Plbd10.72676.04E-773.9525
Susd30.50336.29E-353.9210
Hp0.63338.62E-573.8550
Pmaip10.47673.29E-313.7016
Adgre50.75001.10E-823.5335
Ifi27l2a0.56679.10E-453.3416
Atp1a30.49004.88E-333.3206
Ifitm20.76003.03E-853.2359
Xdh0.48334.07E-323.2003
Pou2f20.47339.21E-313.1216
Rnase60.50672.07E-353.0629
Pla2g70.68002.43E-663.0556
Thbs10.57671.86E-463.0133
Itga40.66003.98E-622.9055
Itgam0.55004.91E-422.7702
Napsa0.60001.49E-502.6112
Sorl10.50336.29E-352.5328
Wfdc170.49675.64E-342.5299
Clec4a10.53006.81E-392.4922
Slc11a10.52672.20E-382.4649
Fyb0.64004.28E-582.4454
Srgn0.66338.14E-632.2151
Cnn20.49331.67E-332.1880
Samhd10.61671.30E-532.1715
Lst10.66671.65E-632.1340
Cd300a0.51006.78E-362.1007
Nr4a10.61002.24E-522.0001
Table 6
Top 20 differentially expressed genes in the Mig-DC vs cDC2 cluster.

ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 20 differentially expressed genes are shown for the migDC vs cDC2 cluster; n = 2 mice.

Mig-DC versus cDC2 cluster top 20 differentially expressed genes
Gene nameStatisticP. valueLog2 fold change
Ccl51.00003.45E-1316.9849
Il12b0.78576.97E-0715.3906
Fscn11.00003.45E-1314.5028
Nudt170.92862.48E-1013.7990
Zmynd150.92862.48E-1013.5523
Il15ra0.92862.48E-1013.5174
Cacnb31.00003.45E-1313.4891
Cd63-ps1.00003.45E-1313.4587
H2-M20.85712.06E-0813.4381
Tnfrsf40.71431.31E-0513.4073
Serpinb6b0.92862.48E-1013.2991
AW1120100.85712.06E-0813.1876
Laptm4b0.85712.06E-0812.9752
Lad10.71431.31E-0512.8948
Cd631.00003.45E-1312.8680
Dnase1l30.71431.31E-0512.7389
Apol7c0.50007.75E-0312.7302
Tm4sf50.50007.75E-0312.6086
Spint20.92862.48E-1012.2644
Zfp8720.64291.57E-0411.4802
Table 7
Top 20 differentially expressed genes in the Mig-DC vs cDC1 cluster.

ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 20 differentially expressed genes are shown for the mig-DC vs cDC1 cluster; n = 2 mice.

Mig-DC versus cDC1 cluster top 20 differentially expressed genes
Gene nameStatisticP. valueLog2 fold change
H2-M20.85712.34E-0914.0574
Tnfrsf40.71433.71E-0613.6282
Zfp8720.64295.96E-0511.348
Arc0.71433.71E-0610.9369
Gbp80.64295.96E-0510.6692
Ramp30.54.51E-0310.6263
Insl60.64295.96E-0510.5787
Gbp40.92861.19E-1110.4833
Adcy60.82012.13E-0810.4176
Atxn10.71433.71E-0610.3352
Ankrd33b0.92861.19E-1110.1242
Dscaml10.64295.96E-059.8945
Src0.54.51E-039.7618
Tmem150c0.64295.96E-059.566
Slc4a80.71164.01E-069.5518
Nudt170.91016.10E-119.5137
Il21r0.69587.81E-069.3355
Fas0.62431.12E-049.3023
Clec2i0.54.51E-039.2653
Cacnb30.98152.44E-138.6751
Table 8
Top 20 differentially expressed genes in the cDC2 vs cDC1 cluster.

ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 20 differentially expressed genes are shown for the cDC2 vs cDC1 cluster; n = 2 mice.

cDC2 versus cDC1 cluster top 20 differentially expressed genes
Gene nameStatisticP. valueLog2 fold change
Mgl20.78575.55E-1512.5236
Cd209d0.54764.76E-0712.1889
Cd209b0.33337.77E-0312.1445
Ms4a6d0.57141.14E-0711.7772
Ms4a4c0.54764.76E-0711.2462
Plaur0.38101.42E-0311.0707
Casp40.38101.42E-0310.0788
Il1rl10.61905.17E-0910.0357
Cd300lg0.33337.77E-039.9463
Cd330.47622.21E-059.7976
Emilin20.40485.52E-049.5336
Il1rl20.38101.42E-039.5332
Creb50.39159.39E-049.2747
Cd209c0.47622.21E-059.0161
Cd209a0.95773.33E-169.0160
Ccl90.67727.43E-119.0010
Fcrls0.38101.42E-038.9226
Il18rap0.39159.39E-048.7118
Wfdc170.72491.46E-128.5219
Tent5a0.65344.52E-108.2999
Table 9
Top 25 differentially expressed genes in the mast cell cluster.

ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 25 upregulated genes are shown for the mast cell cluster; n = 2 mice.

Mast cell top 25 differentially expressed genes
Gene nameStatisticP. valueLog2 fold change
Tpsb21.00008.9E-1618.7534
Mcpt40.87502.4E-0717.4253
Tpsab10.87502.4E-0715.5899
Cma11.00008.9E-1615.0710
Slc45a30.75005.2E-0511.7029
Rprm0.87502.4E-0711.6969
Hs6st20.75005.2E-0511.1665
Maob0.86833.4E-0710.7834
Cpa30.99001.8E-1310.6394
Ednra0.75005.2E-0510.2131
Gata10.75005.2E-0510.0639
Kit1.00008.9E-169.2771
Mlph0.74675.9E-058.5668
Cma20.75005.2E-058.5455
Cyp11a10.99334.9E-148.4308
Tph10.87172.8E-078.3091
Rab27b0.87172.8E-078.1436
Il1rl10.97331.4E-117.7399
Itga2b0.83501.8E-067.6658
Gata20.99334.9E-147.5597
Poln0.73678.2E-057.2024
Slc18a20.96001.4E-106.8570
Stard130.74675.9E-056.7772
Adora30.70332.3E-046.4180
Kcne30.80506.6E-066.4033
Table 10
Top 25 differentially expressed genes in the basophil cluster.

ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 25 upregulated genes are shown for the basophil cluster; n = 2 mice.

Basophil top 25 differentially expressed genes
Gene nameStatisticP. valueLog2 fold change
Mcpt80.73472.22E-1615.6663
Ms4a20.86762.22E-1612.3460
Gata20.90842.22E-169.7841
Il40.68722.22E-168.7980
Cd200r30.95252.22E-168.6767
Il60.83092.22E-168.5795
Ccl40.90212.22E-167.0748
Ifitm10.93632.22E-166.8128
Cyp11a10.97292.22E-166.6271
Ccl30.92252.22E-165.9632
Hgf0.73262.22E-165.7653
Csf10.89212.22E-165.4838
Ccl90.88252.22E-165.3108
Ifitm70.75592.22E-165.1284
Aqp90.75632.22E-165.0857
Rab440.65342.22E-165.0335
Hdc0.91332.22E-164.9976
Cd690.74542.22E-164.9828
Cdh10.71972.22E-164.9643
Il18r10.65392.22E-164.5920
Csf2rb20.69302.22E-164.3472
Lilr4b0.87882.22E-164.1383
Rgs10.70012.22E-164.0712
Il18rap0.75712.22E-164.0030
Osm0.65592.22E-163.6933
Table 11
Top 25 differentially expressed genes in the neutrophil cluster.

ScRNA-Seq was performed on live CD45+ lung cells isolated from E18.5, P1, P7 and P21 B6 pups. Top 25 upregulated genes are shown for the neutrophil cluster; n = 2 mice.

Neutrophil top 25 differentially expressed genes
Gene nameStatisticP. valueLog2 fold change
Retnlg0.78331.9E-9116.7841
Stfa20.90671.6E-13113.3347
BC1005300.90333.1E-13011.5407
Mmp90.88675.5E-12411.2071
Asprv10.75001.1E-8210.6669
Stfa2l10.93004.5E-1418.5427
S100a90.95672.7E-1538.3546
2010005H15Rik0.84672.9E-1108.1305
BC1170900.88001.4E-1217.8635
Slpi0.87672.1E-1207.5069
Stfa10.89671.1E-1276.8526
Il1r20.71338.4E-746.6465
S100a80.94671.6E-1486.2103
Slc7a110.69332.9E-695.9640
Csf3r0.74003.5E-805.7411
Cxcr20.74335.2E-815.6599
Wfdc210.77339.5E-895.3370
Pglyrp10.83674.4E-1074.9416
Mxd10.68002.4E-664.5560
Hdc0.82675.5E-1044.2867
Grina0.70331.7E-714.0795
Il1b0.65671.9E-613.8434
Thbs10.67336.5E-652.7913
S100a110.76003.0E-852.6263
Srgn0.66004.0E-622.3914
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background (M. musculus)C57BL/6JCharles Rivers Laboratories
Antibodyanti-CD3e (Hamster monoclonal)BD BiosciencesCat# 553062; RRID:AB_394595FACS (1:100)
Antibodyanti-CD4 (Rat monoclonal)BD BiosciencesCat# 552051; RRID:AB_394331FACS (1:100)
Antibodyanti- CD8a (Rat monoclonal)BioLegendCat# 100721; RRID:AB_312760FACS (1:100)
Antibodyanti-Tbet (Mouse monoclonal)BioLegendCat# 644808; RRID:AB_15955479FACS (1:100)
Antibodyanti-GATA3 (Mouse monoclonal)BD BiosciencesCat# 563510; RRID:AB_2738248FACS (1:100)
Antibodyanti-Foxp3 (Rat monoclonal)eBioscienceCat# 12-5773-82; RRID:AB_465936FACS (1:100)
Antibodyanti-Rorγt (Mouse monoclonal)BD BiosciencesCat# 562607; RRID:AB_11153137FACS (1:100)
Antibodyanti-IFNγ (Rat monoclonal)BioLegendCat# 505829; RRID:AB_10897937FACS (1:100)
Antibodyanti-IL-4 (Rat monoclonal)BD BiosciencesCat# 562045; RRID:AB_10895799FACS (1:100)
AntibodyIL-10 (Rat monoclonal)BioLegendCat# 505027; RRID:AB_2561522FACS (1:100)
AntibodyIL-17 (Rat monoclonal)Miltenyi BiotecCat# 130-095-732; RRID:AB_10828821FACS (1:100)
Commercial assay or kitFISH probe: anti-mouse Plac8Advanced Cell Diagnostics (ADC)Cat# 532701RNAscope: 50 μl per slide
Commercial assay or kitFISH probe: anti-mouse Dab2ADCCat# 558131-C2RNAscope: (1 μl per 50 μl)
Commercial assay or kitFISH probe: anti-mouse Cd68ADCCat# 316611-C2RNAscope: (1 μl per 50 μl)
Commercial assay or kitFISH probe: anti-mouse-Cd68ADCCat# 316611-C3RNAscope: (1 μl per 50 μl)
Commercial assay or kitFISH probe: anti-mouse-Cd68ADCCat# 316611RNAscope: 50 μl per slide
Commercial assay or kitFISH probe: anti-mouse-Cdh5ADCCat# 312531-C2RNAscope: (1 μl per 50 μl)
Commercial assay or kitFISH probe: anti-mouse-Mki67ADCCat# 416771-C2RNAscope: (1 μl per 50 μl)
Commercial assay or kitFISH probe: anti-mouse-Car4ADCCat# 468421-C2RNAscope: (1 μl per 50 μl)
Commercial assay or kitFISH probe: anti-mouse-GalADCCat# 400961-C2RNAscope: (1 μl per 50 μl)
Commercial assay or kitFISH probe: anti-mouse-C1qaADCCat# 441221RNAscope: 50 μl per slide
Commercial assay or kitFISH probe: anti-mouse-EpcamADCCat# 418151-C2RNAscope: (1 μl per 50 μl)
Commercial assay or kitFISH probe: anti-mouse-ItgaxADCCat# 311501-C3RNAscope: (1 μl per 50 μl)
Commercial assay or kitFISH probe: anti-mouse-Ifitm6ADCCat# 511321-C3RNAscope: (1 μl per 50 μl)
Commercial assay or kitFISH probe: anti-mouse-Lair1ADCCat# 509151RNAscope: 50 μl per slide
Commercial assay or kitFISH probe: anti-mouse-Ccr2ADCCat# 501681RNAscope: 50 μl per slide

Data availability

Sequencing data have been deposited in GEO under accession code GSE147668. Gene count and metadata tables are also available on FigShare at https://figshare.com/articles/Diverse_homeostatic_and_immunomodulatory _roles_of_immune_cells_in_the_developing_mouse_lung_revealed_at_single_cell_resolution/12043365.

The following data sets were generated
  1. 1
    NCBI Gene Expression Omnibus
    1. R Domingo-Gonzalez
    2. ZaniniF
    3. X Che
    4. M Liu
    5. RC Jones
    6. MA Swift
    7. SR Quake
    8. DN Cornfield
    9. CM Alvira
    (2020)
    ID GSE147668. Diverse homeostatic and immunomodulatory roles of immune cells in the developing mouse lung revealed at single cell resolution.
The following previously published data sets were used
  1. 1
    ArrayExpress
    1. J Schyns
    2. Q Bai
    3. C Ruscitti
    4. C Radermecker
    5. S De Schepper
    6. S Chakarov
    7. D Pirottin
    8. F Ginhoux
    9. G Boeckxstaens
    10. F Bureau
    11. T Marichal
    (2019)
    ID 10.1038/s41467-019-11843-0. scRNA-seq analysis of lung CD64-expressing mononuclear cells, patrolling and classical monocytes from steady-state C57BL/6J mice.
  2. 2
    FigShare
    1. Tabula Muris Consortium
    (2018)
    ID 10.1038/s41586-018-0590-4. Tabula Muris: Transcriptomic characterization of 20 organs and tissues from Mus musculus at single cell resolution: Single-cell RNA-seq data from Smart-seq2 sequencing of FACS sorted cells (v2).

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