Corrected 10 April 2019; see below

www.sciencemag.org/content/363/6432/eaau0964/suppl/DC1

Supplementary Materials for

Two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches

Svetoslav Chakarov, Hwee Ying Lim, Leonard Tan, Sheau Yng Lim, Peter See, Josephine Lum,

Xiao-Meng Zhang, Shihui Foo, Satoshi Nakamizo, Kaibo Duan, Wan Ting Kong, Rebecca Gentek, Akhila Balachander, Daniel Carbajo, Camille Bleriot, Benoit Malleret,

John Kit Chung Tam, Sonia Baig, Muhammad Shabeer, Sue-Anne Ee Shiow Toh, Andreas Schlitzer, Anis Larbi, Thomas Marichal, Bernard Malissen, Jinmiao Chen,

Michael Poidinger, Kenji Kabashima, Marc Bajenoff, Lai Guan Ng, Veronique Angeli, Florent Ginhoux*

*Corresponding author. Email: florent_ginhoux@immunol.a-star.edu.sg

Published 15 March 2019, Science 363, eaau0964 (2019)

DOI: 10.1126/science.aau0964

This PDF file includes:

Materials and Methods Figs. S1 to S7 Captions for Tables S1 to S5 Captions for Movies S1 and S2

Other Supporting Online Material for this manuscript includes the following: (available at www.sciencemag.org/content/363/6432/eaau0964/suppl/DC1)

Tables S1 to S5 (Excel) Movies S1 and S2

Correction: Fig. S1 has been updated to correct a conversion error that resulted in some parts of the figure not being visible.

Materials and Methods REAGENT or RESOURCE SOURCE IDENTIFIER Mouse Antibodies Rat anti-mouse CD19, APC Biolegend Cat#115512 Armenian hamster (AH) anti-mouse CD3, APC Biolegend Cat#100312 Rat anti-mouse CD49b, APC Biolegend Cat# 108910 Rat anti-mouse Ly6G, APC Biolegend Cat# 127614 Rat anti-mouse CD45, APC/Cy7 Biolegend Cat#103115 Rat anti-mouse CD45, BUV395 BD Bioscience Cat#564279 Mouse anti-mouse CD64, PE BD Bioscience Cat#55845 Mouse anti-mouse CD64, BV711 Biolegend Cat# 139311 Goat anti-mouse MerTK, Biotin RnD Systems Cat# BAF591 Rat anti-mouse MerTK, PE eBioscience Cat# 12-5751-82 Rat anti-mouse MerTK, PE/Cy7 eBioscience Cat# 25-5751-82 Rat anti-mouse SiglecF, BV421 BD Bioscience Cat# 562681 Rat anti-mouse SiglecF, PE BD Bioscience Cat# 552126 Rat anti-mouse CD11b, BV650 BD Bioscience Cat# 563402 AH anti-mouse CD11c, BV605 Biolegend Cat# 117333 Rat anti-mouse Ly6C, APC/Cy7 Biolegend Cat# 128026 Rat anti-mouse Ly6C, APC eBioscience Cat# 17-5932-82 Rat anti-mouse Ly6C, PerCP/Cy5.5 eBioscience Cat# 45-5932-82 Rat anti-mouse F4/80, eFluor 450 eBioscience Cat# 48-4801-82 Rat anti-mouse F4/80, PE/CF594 BD Bioscience Cat# 565613 Rat anti-mouse F4/80, APC/Cy7 BD Bioscience Cat# 47-4801-80 Mouse anti-mouse CX3CR1, PE Biolegend Cat# 149006 Rat anti-mouse EpCAM, PE/Cy7 Biolegend Cat# 118216 Rat anti-mouse EpCAM, BV605 Biolegend Cat# 118227 Rat anti-mouse Lyve1, AF488 eBioscience Cat# 53-0443 Rat anti-mouse Lyve1, eFluor 660 eBioscience Cat# 50-0443 Rat anti-mouse CD44, APC/eFluor 780 eBioscience Cat# 47-0441 Rat anti-mouse Clec7a, PE BD Bioscience Cat# 144303 Rat anti-mouse CD72, PE BD Bioscience Cat# 550269 Rat anti-mouse CD38, APC eBioscience Cat# 17-0381 Rat anti-mouse Mrc-1, AF647 Biolegend Cat# 141712 AH anti-mouse CD36, APC Biolegend Cat# 102611 Rat anti-mouse CD115, PerCP/efluor 710 eBioscience Cat# 46-1152-80 Rat anti-mouse CD88, PE/Cy7 Biolegend Cat# 135809 Mouse anti-mouse CD45.1, PE/Cy7 eBioscience Cat# 25-0453-82 Mouse anti-mouse CD45.2, PerCP/Cy5.5 eBioscience Cat# 45-0454-82 Rat anti-mouse MHCII, AF700 Biolegend Cat# 107622 AH anti-mouse CD3, PE/Cy7 eBioscience Cat# 25-0031-82 AH anti-mouse CD3, PerCP/efluor 710 eBioscience Cat# 46-0032-82 Rat anti-mouse CD19, Pacific Blue eBioscience Cat# 57-0193-82 Rat anti-mouse Ly6G, PE-CF594 BD Bioscience Cat# 562700

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Rat anti-mouse Ly6G, PE-CF594 BD Bioscience Cat# 562700 Rat anti-mouse Foxp3, PE-CF594 BD Bioscience Cat# 562466 Rat anti-mouse ZBTB46, PE BD Bioscience Cat# 565832 Rat anti-mouse CD206, AlexaFluor647 Biolegend Cat# 141712 Rat anti-mouse CD4, PE/Cy7 eBioscience Cat# 25-0042 Rat anti-mouse CD169, PE Biolegend Cat# 142403 Rat anti-mouse CD62L, eFluor450 eBioscience Cat# 48-0621 Human Antibodies Mouse anti-human CD45, AlexaFluor488 Biolegend Cat# 304019 Mouse anti-human CD45, BUV395 BD Bioscence Cat# 563792 Mouse anti-human CD16, PerCP-eFluor 710 eBioscience Cat# 46-0168-42 Mouse anti-human CD206, APC Biolegend Cat# 321109 Mouse anti-human CD14, APC-Cy7 Biolegend Cat# 325620 Mouse anti-human CD64, PE Biolegend Cat# 305008 Mouse anti-human CD64, BV711 BD Bioscence Cat# 740782 Goat anti-human Lyve1, Biotin R&D Cat# BAF2089 Mouse anti-human CD163, BV605 BD Bioscence Cat# 745091 Rat anti-mouse/human CD11b, BV655 Biolegend Cat# 101259 Mouse anti-human HLA-DR, AlexaFluor700 Biolegend Cat# 327014 Mouse anti-human CD169, FITC Miltenyi Biotec Cat# 130-098-656 Mouse anti-human CD169, BUV395 BD Bioscence Cat# 742997 Chemicals, Peptides and Recombinant Protein Collagenase type IV Sigma-Aldrich Cat# C5138 DNase I Roche Cat#10104159001 Fetal Bovine Serum GIBCO Cat#10270-106 RPMI eBioscience Cat#00433357 Tamoxifen Sigma-Aldrich Cat#T5648 eBioscience™ Foxp3 Kit eBioscience Cat# 00-5521-00 Isoprenaline hydrochloride Sigma-Aldrich Cat# I5627

Mice.

C57BL/6 (Cd45.2+) mice were from the Biological Resource Center, Agency for Science, Technology and Research, Singapore. Congenic C57BL/6 (Cd45.1+), Cx3cr1gfp/+ and Ccr2tm1Ifc mice were from the Jackson Laboratory. S100a4Cre/WT[BALB/c-Tg(S100a4Cre)1Egn/YunkJ] were purchased from the Jackson Laboratory and bred in house with RosaR26R-EYFP/R26R-EYFP mice. Generation of Slco2b1-IRES-LoxP-STOP-LoxP-hDTR (called Slco2b1flox/DTR here) gene-targeted mice is described below. Lyve1tm1.1(EGFP/cre)Cys were purchased from the Jackson Laboratory and in bred with Slco2b1flox/DTRor Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze mice. LyzMtm1(cre)Ifo (LyzMcre) were from the Jackson Laboratory and bred with Slco2b1flox/DTR.

Cdh5CreERRosatdTomato and Wnt1Cre-RosatdTomato were kindly provided by Dr Marc Bajenoff and bred with Cx3cr1GFP/+ in CIML Marseille France. Mafbtm(GFP)UTR mice were kindly provided by S. Takahashi, Tsukuba University, Japan. Csf1rMer-iCre-Mer1Jwp (Csf1rMerCreMer) were from the Jackson Laboratory and were bred and crossed in house with B6;129S6-Gt(ROSA)26Sortm9(CAG-

tdTomato)Hze/J kindly provided by Dr Veronique Angeli.

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B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze were kindly provided by Dr N Barker, Institute of Medical Biology (IMB) Singapore

Bone marrow chimeras were realized using recipient Cd45.1+ C57BL/6 mice lethally irradiated (2x 600 rad, 3 hr apart using a Cesium source) and reconstituted with CD45.2+ BM isolated from Slco2b1flox/DTR, Lyve1Cre-Slco2b1flox/DTR or ITIB mice. Engraftment was assessed by measuring the % of donor cells among blood Ly6C+ and LyG+ granulocytes 4 weeks after transplantation. Mice were analyzed two to tree months post-transplant.

Parabiotic mice were generated by suturing age- and weight-matched WT CD45.1+ and Ccr2-/- Cd45.2+ mice at the age of 6 weeks. Mice were analyzed at the indicated age point.

For tamoxifen treatment mice were fed with 500mg/kg tamoxifen supplemented diet (Envigo) for 7 days and analyzed at the indicated time point after the end of the diet.

All mice were bred and maintained in the Singapore Immunology Network animal facility before use at 5-10 weeks of age or indicated age point. Only healthy male mice were used in this study. All experiments and procedures were approved by the Institutional Animal Care and Use Committee of the Biological Resource Center (Agency for Science, Technology and Research, Singapore) in accordance with the guidelines of the Agri-Food and Veterinary Authority and the National Advisory Committee for Laboratory Animal Research of Singapore. Preparation of cell suspensions.

All mouse and human organs were digested for 30 min in RPMI 10% FBS and collagenase type IV (0.2 mg/ml; working activity of 770 U/mg) (Sigma). For sorting, tissue cell suspensions underwent preenrichment for CD45+ cells with mouse CD45 MicroBeads, (Miltenyi) and were separated on an AutoMacs (Miltenyi). OTII Proliferation Assay

Lung AM, DC, Lyve1hiMCHIIlo and Lyve1loMCHIIhi IM were FACS-sorted from lungs of WT mice. OTII T cells were negatively selected from the spleens of OTII-Rag1-/- mice using the CD4+ T cell enrichment kit (Miltenyi) and were separated on an AutoMacs (Miltenyi). Enriched T cells were washed with PBS, and labeled with 1uM CFSE (Invitrogen). DCs and T-cells were cultured for 72h at a ratio of 1:20 and proliferation was analyzed using flow cytometry and intracellular Foxp3 staining kit Mouse treatment and examination of fibrosis For lung fibrosis mice were anaesthetized with Ketamine/Xylazine during bleomycin administration. Bleomycin (0.5µg per g body weight) was administrated to the mice intratrnasally. For heart fibrosis, mice were treated with 25µg/g of isoprenaline in subcutaneous for 5 consecutive days. In some case Lyve1hiMHCIIlo IM were depleted in Lyve1-SLCO mice using 1µg/200µl DTx. DTx was administered intraperitoneally (i.p.) 16 hours before bleomycin treatment for lung fibrosis or at day 5 of isoprenaline treatment for hert fibrosis. Mice were also monitored for bleomycin-induced and isoprenaline-induced weight lost every day post treatment.

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Fig. S1.

(A) Flow cytometric plots showing gating strategy for lung IM single-cell RNA sequencing sort. (B) ‘Violin’ plots showing gene numbers (left), percentage of mitochondrial genes (middle) and read number (nUMR) (right) detected per cell. Two cells were defined as outliers and shown in blue: one with mitochondrial gene percentage > 0.1 and another one with a UMI number < 9.8×105.

A

SS

C-A

FCS-A

FCS

-W

FCS-H

DA

PI

SSC-A

Lin

CD45

DAPI-CD45+

Mer

TK

CD64

Lin-CD45+

Sig

lecF

CD11b

MerTK+CD64+

20.0%

40.0%

5.0%

84.0%98.0%72.0%

3

2

4

Gen

e nu

mbe

r ×10

3

B

Fig. S1.

Mito

chon

dria

l gen

es (%

)

0.050

0.075

0.100

0.025

0.000

0.125

UM

I num

ber ×

105

9.8

9.9

10.0

9.7

9.6

5

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Fig. S2.

(A) Significant biological processes or pathways identified using David software between cluster 1 and cluster 2 using DEGs identified in single-cell analysis in Fig1A-C. The bar plot indicates the top seven upregulated pathways in cluster 1 (blue) or cluster 2 (red). (B) CMap analysis of single lung IMs (from Fig1A-C) showing their enrichment for Lyve1loMHCIIhi and Lyve1hiMHCIIlo macrophage gene sets defined from bulk RNAseq in Fig1D-F. (C) PC analysis of gene expression of sorted AMs, lung monocytes, MHCII− and MHCII+ IMs. The 44 genes with the highest –log10(P value) and log2(fold change) were plotted as a heat map in (D). (E) Flow cytometric measurement of marker expression on Lyve1loMHCIIhi and Lyve1hiMHCIIlo IMs in Fig1, from wild-type or transgenic MafGFP mice. (F) Flow cytometric measurement of ZBTB46 expression in lung monocytes, AMs, Lyve1loMHCIIhi and Lyve1hiMHCIIlo IMs, cDC1 and cDC2 from wild-type mice. (G) Experimental outline for tamoxifen feeding of Csf1rMerCreMer-RosatdTomato mice (upper panel) and CSF1R-tdTomato expression measurement by flow cytometry in lung monocytes, AMs, Lyve1loMHCIIhi and Lyve1hiMHCIIlo IMs (lower panel). (H) S100a4 gene expression detected by RNAseq in lung monocytes, AMs, Lyve1loMHCIIhi and Lyve1hiMHCIIlo

IMs. (I) Representative flow cytometric plots showing the relative frequencies of CD45.1+ and CD45.2+ cells within blood CD3+ T cell (left panel), Ly6Chi and LyC6lo blood monocytes (right panel) from the Ccr2-/- Cd45.2+ parabiont (upper panel) after 6 months of surgical attachment to WT Cd45.1+ counter partner (lower panel). Bars represent mean ± SEM (ns not significant; * P ≤ 0.1; ****P ≤ 0.0001).

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Fig. S3.

(A) Slco2b1 gene expression detected in IMMGEN data from sorted small intestine (SI), lung, brain, lung macrophages; bone marrow (BM) and blood monocytes; lung and spleen DC. (B) RNAseq quantification of Slco2b1 gene expression in FACS-purified Lyve1hiMHCIIlo and Lyve1loMHCIIhi IMs, alveolar macrophages (AM) and lung monocytes (Lg Mono.) from indicated tissues of WT C57Bl/6 mice. The log2(RPKM)values are shown. (C) Flow cytometry measurement of intracellular SLCO2B1 expression in lung Lyve1hiMHCIIlo and Lyve1loMHCIIhi IMs, AM and monocytes from WT C57Bl/6 mice (left panel); geometric mean (GM) ±SEM SLCO2B1 expression in the indicated populations (right panel). (Iso. = Isotype control) (D) Schematic representation of Slco2b1flox/DTR construct. (E) Mean cell number of indicated immune cell populations from lungs of LyzMCre-Slco2b1flox/DTR mice either not treated or 24 hours after DTx treatment. (F) Mean cell number of AM and IM from lungs of LyzMCre-Slco2b1flox/DTR mice either not treated or 24 hours after low-dose DTx treatment. (G) Flow cytometry analysis of percentage of CD45.1+ AM and Lyve1loMHCIIhi and Lyve1hiMHCIIlo IMs in low-dose DTx-treated Cd45.2+ LyzMCre-Slco2b1flox/DTR mice 9 days after adoptive transfer of CD45.1+ Ly6Chi bone marrow monocytes (left panels); mean percentage ± SEM of CD45.1+ cells within Lyve1hiMHCIIlo and Lyve1loMHCIIhi IM populations (right panels). Bars represent mean ± SEM (*P ≤ 0.05; ** P ≤ 0.01; **** P ≤ 0.0001).

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Fig. S4.

(A) Volcano plots of relative difference in expression level of DEGs between 29 Lyve1loMHCIIhi and 34 Lyve1hiMHCIIlo IM-related AVM macrophages identified by cMAP in (Fig. 3A lower panel). -log10(P value) ≥ 1.5 DEGs are shown in blue; -log10(P value) ≥ 3 and -1 ≥ log2(fold change) ≤ 1 DEGs are shown in red; genes of interest are highlighted in yellow. (B) cMap analysis of single bladder, lung, mammary gland, muscle, pancreas and uterus macrophages showing their enrichment for Lyve1loMHCIIhi and Lyve1hiMHCIIlo gene sets found in (Fig. 3D). (C) Volcano plots of relative difference in expression level of DEGs between Lyve1loMHCIIhi and Lyve1hiMHCIIlo IM-related bladder, lung, mammary gland, muscle, pancreas and uterus macrophages identified by cMAP in (B). -log10(P value) ≥ 3 DEGs are shown in blue; -log10(P value) ≥ 3 and -0.5 ≥ log2(fold change) ≤ 0.5 DEGs are shown in red; genes of interest are highlighted in yellow. (D) Flow cytometry analysis of Lyve1hiMHCIIlo macrophages from the indicated tissues of WT mice (E) Geometric mean (GM) forward scatter-A (FSC), indicating cell size, of Lyve1loMHCIIhi and Lyve1hiMHCIIlo macrophages from heart, fat and skin of WT mice. (F) Volcano plots of relative difference in expression level of DEGs between Lyve1loMHCIIhi and Lyve1hiMHCIIlo macrophages identified in (Fig 3D): -log10(P value) ≥ 3 DEGs are shown in blue; -log10(P value) ≥ 4 and -2 ≥ log2(fold change) ≤ 2 DEGs are shown in red; genes of interest are highlighted in yellow. (G) Significant processes, networks and pathways identified by genego (https://portal.genego.com) using a p-value ≥ 0.0001 and log2(fold change) ≥ 1.5 DEGs between Lyve1loMHCIIhi and Lyve1hiMHCIIlo macrophages. (H) Seurat tSNE analysis of myeloid cells from non-malignant human lung defined 5 clusters of cross presenting DC, granulocytes, langerhans cells, macrophages and monocyte-derived DCs. (I) Seurat tSNE analysis of the macrophage cluster defined in (H). Cells with positive (red) and negative (blue) cMAP score were projected on the tSNE with zero (green) cMAP score (upper panel) and without zero cMAP score (lower panel). (J) Flow cytometric analysis of Lyve1 and CD206 expression in human macrophage in lung from three different donors (left panel) or in omental and subcutaneous fat from a single human donor. Bars represent mean ± SEM (ns not significant; * ≤ 0.01; ** P ≤ 0.01; *** P ≤0.001; **** P ≤0.0001).

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Fig. S5.

(A) Immunofluorescence microscopy images of 250-µm-thick lung, fat, heart sections or whole mount ear skin from Cdh5creER-RosatdTomato mice treated with tamoxifen (5 mg in corn oil, 200-µl gavage) in order to induce tdTomato expression in all endothelial cells (red); Lyve1hiMHCIIlo cells are visualized with anti-Lyve1 mAb (white) and macrophages using anti-Iba1 mAb (cyan). White arrows indicate Lyve1hiMHCIIlo macrophages surrounding blood vessels. Scale bars represent 40 µm. (B) Laser confocal microscopy images of 250-µm-thick lung, fat, or heart sections or whole-mount ear skin from Wnt1Cre-RosatdTomato-Cx3cr1GFP/+ mice showing Lyve1loMHCIIhi

macrophages (green) closely associated with sympathetic nerve bundles (Wnt1+) (red). Scale bars represent 50 µm.

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Fig. S6.

(A) Mean expression level of Il10 in FACS-purified lung monocytes (Lg Mono), alveolar macrophages (AM), and Lyve1loMHCIIhi and Lyve1hiMHCIIlo IM from WT mice, measured by bulk RNA sequencing. (B) Flow cytometric analysis of the frequency of IL-10-producing Lyve1loMHCIIhi and Lyve1hiMHCIIlo lung IMs in IL-10-β-lactamase reporter mice (left panels). (B, C) Graphs show the mean percentage of IL-10-producing IMs in lung (B right panel) or heart, fat and skin (C). (D) Flow cytometric plots showing proliferation of CFSE-labeled OTII CD4+ T cells after 3 days co-culture with ovalbumin-pulsed lung alveolar macrophages (AM), dendritic cells (DC), or Lyve1loMHCIIhi, and Lyve1hiMHCIIlo IMs (left panels); mean percentage of proliferating cells in cultures of T cells alone, or T cells cultured with the cell populations in (D) (right panels). (E) Flow cytometric analysis of the frequency of intracellular FoxP3 expression in proliferating OTII CD4+ T cells from (D) (left panels); mean percentage of FoxP3-expressing T cells in co-cultures of T cells with DC, or Lyve1loMHCIIhi and Lyve1hiMHCIIlo IMs (right panel). A representative plot of three independent experiments is shown. (*P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).

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Fig. S7.

(A) Gene set enrichment analysis of RNAseq expression levels of genes involved in response to wound-healing response genes (GO: GO:0009611) in Lyve1loMHCIIhi and Lyve1hiMHCIIlo IMs from lung (top panel), and from the lung, heart, fat, and skin combined (bottom panels). (B) Flow cytometric measurement of Lyve1hiMHCIIlo IM frequency in tissues from wild-type Cd45.1+ WT mice reconstituted with BM from Cd45.2+ Lyve1Cre/GFP (Lyve1→CD45.1) (top row) or Cd45.2+ Lyve1cre-Slco2b1flox/DTR (Lyve1-SLCO→CD45.1) mice (bottom row) and treated i.p. with 1 µg/200 µl DTx 2 months after reconstitution (left panels); mean percentage of Lyve1hiMHCIIlo IMs within all live hematopoietic cells (CD45+) in the indicated tissues (right panels). The chimeras were analyzed 16 hours after DTx treatment. (C) Experimental outline for bleomycin-induced lung fibrosis and DTx treatment. (D) Hematoxylin and eosin (H&E) (top panels) and trichrome (TRI) (bottom panels) staining of lung sections from Lyve1→ Cd45.1+ and Lyve1-SLC→ Cd45.1+ chimeric mice 14 days after DTx and bleomycin treatment. Scale bar = 100 µm (E) Experimental outline for isoproterenol-induced heart fibrosis and DTx treatment (s.c. = subcutaneous). (F) Mean weight loss during isoproterenol treatment as a percentage of day 0 weight. (G) Ratio between heart weight and total weight, measured on day 19 of isoproterenol treatment. (H) Mean number of Ly6G+ neutrophils and F4/80+CD64+ inflammatory cells in heart of isoproterenol-treated mice at day 19 measured by flow cytometry. Bars represent mean ± SEM (*P ≤ 0.05; ** P ≤ 0.01).

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Table S1. Number of expressed genes in individual cell for Figure S1B.

Table S2. Gene signatures of lung Lyve1loMHCIIhi IMs used in Figure 3A.

Table S3. Gene signatures of lung Lyve1hiMHCIIlo IMs used in Figure 3A.

Table S4. Gene signatures of lung Lyve1loMHCIIhi IMs and mouse orthologs of human transcripts used in Figure 3E.

Table S5. Gene signatures of lung Lyve1hiMHCIIlo IMs and mouse orthologs of human transcripts used in Figure 3E.

Movies S1 and S2. WT Cd45.1+ mice were irradiated and reconstituted with bone marrow from either control Lyve1cre/GFP (Movie S1) or Lyve1cre/GFP-Slco2b1flox/DTR (Movie S2) mice. Two months later, they were treated with 1µg/20µL DTx to deplete Lyve1hiMHCIIlo cells, followed by intravenous transfer of 5×106 Ly6Chi inflammatory monocytes purified from ubi-GFP mice. Animals were analyzed 16 hours after transfer. A representative movie of four mice experiment is shown. Three independent experiments were performed.

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