1. Volume 44, Issue 4, Pages 755-768 (April 2016)
Yolk Sac Macrophages, Fetal Liver, and Adult Monocytes Can Colonize an Empty Niche and Develop into Functional Tissue-Resident Macrophages 
Lianne van de Laar, Wouter Saelens, Sofie De Prijck, Liesbet Martens, Charlotte L. Scott, Gert Van Isterdael, Eik Hoffmann, Rudi Beyaert, Yvan Saeys, Bart N. Lambrecht, Martin Guilliams 
Immunity 
Volume 44, Issue 4, Pages (April 2016)
DOI: /j.immuni
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Figure 1
YS-Macs, FL-MOs, and BM-MOs Have Distinct Gene Expression Profiles
To visualize differential gene expression between CD45.1+CD45.2+ E12.5 YS-Macs, E15.5 FL-MOs, and week 6–12 BM-MOs (see Figure S1 for gating strategies), each gene was plotted in a hexagonal diagram in which the direction of a point represents an upregulation in one or two populations and the distance from the origin represents the strength of this upregulation. Genes that are ≥32-fold differentially expressed are plotted on the outer grid line. Rose diagrams (right upper corner of each hexagon) show the percentages of genes in each orientation.
(A) Hexagon and rose diagram of all arrayed genes. Ccr2, Mertk, Siglecf, and Tubb5 are shown as examples. Bar plots show mean ± SEM Log2 expression profiles of the indicated genes.
(B–F) Genes associated with antigen uptake, anti-bacterial defense, cell movement, immune response, lipid metabolism, pattern recognition (B), chemokine recognition (C), anti-viral response (D), cell division and DNA replication (E), and antigen presentation (F). See Table S1 for data on the individual GO terms and gene lists contributing to each module.
(G) Per module, the statistical significance of the mean orientation of the differentially expressed genes was determined. Adjusted p values are plotted in the direction of the circular mean.
Data are obtained from four independent replicates from different experiments. See also Figure S1 and Table S1.
Immunity  , DOI: ( /j.immuni )
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 2
FL-MOs Outcompete YS-Macs and BM-MOs in Colonizing the AM Niche
CD45.1+CD45.2+ YS-Macs, CD45.1+ FL-MOs, and CD45.2+YFP+ BM-MOs (see Figure S2 for gating strategies) were pooled in a 1:1:1 ratio and intranasally transferred into Csf2rb−/− pups. Transfer-derived cells were analyzed in the lungs (week 1) and BAL (weeks 2, 6, 10).
(A) Schematic overview of the experiment.
(B and C) Expression of CD11b, F4/80, CD11c, and SiglecF by transfer-derived cells (see Figure S3 for gating strategy). Representative flow cytometry plots (B) and mean ± SEM mean fluorescence intensity (MFI) (C) are shown.
(D) Number of cells retrieved from each precursor type.
(E and F) Mean ± SEM percentages (E) and representative flow cytometry plots (F) of cells retrieved from each precursor type within the total transfer-derived cell population.
(G) CD116 and CD131 expression on sorted YS-Macs, FL-MOs, and BM-MOs.
(H) Mean ± SEM ΔMFI (MFI stimulated cells − MFI unstimulated cells) of pSTAT5 in YS-Macs, FL-MOs, and BM-MOs cultured for 30 min in the presence of indicated concentrations of GM-CSF (see Figure S4 for histograms).
(I) Mean ± SEM retrieved cells from cultures in the presence of 30 ng/mL GM-CSF started with 20,000 YS-Macs, FL-MOs, or BM-MOs.
A representative experiment (four to five mice per time point in B–F; at least two replicates per condition in G–I) of two independent experiments is shown. Statistical testing compared to FL-MOs. See also Figures S2–S4.
Immunity  , DOI: ( /j.immuni )
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4. Figure 3
YS-Macs, FL-MOs, and BM-MOs All Generate a Significant AM Population
Csf2rb−/− pups were intranasally transferred with YS-Macs, FL-MOs, BM-MOs, BAL-AMs, or PBS as a control and sacrificed 6 weeks or 1 year after transfer. Non-transferred wild-type (WT) mice were sacrificed at 6 weeks or 1 year of age.
(A) Schematic overview of the experiment.
(B) Representative flow cytometry plots showing the phenotype of transfer-derived and WT cells 6 weeks after transfer (autofl = autofluorescence; see Figure S5 for gating strategy).
(C and D) Numbers of WT and transfer-derived F4/80hiCD11chiSiglecFhi AMs in the BAL of WT and recipient Csf2rb−/− mice 6 weeks (C) and 1 year (D) after transfer, shown per mouse and as mean ± SEM per group.
Data were pooled from at least three (B, C) and at least two (D) independent transfer experiments per cell type. 12 controls (5 WT, 7 PBS) were derived from experiments that included conditions presented in Figures 3C and 7C and are therefore shown twice. See also Figure S5.
Immunity  , DOI: ( /j.immuni )
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5. Figure 4
YS-Mac-, FL-MO-, and BM-MO-Derived Cells Have an AM-Specific Gene Expression Profile
Csf2rb−/− pups were intranasally transferred with CD45.1+CD45.2+ YS-Macs or FL-MOs or BM-MOs or BAL-AMs (input cells). Mice were sacrificed 6 weeks after transfer and YS-Mac-, FL-MO-, BM-MO-, and BAL-AM-derived AMs (output cells) were FACS sorted from the BAL as CD45.1+CD45.2+F4/80+CD11chiSiglecFhi cells. AMs from 6-week-old non-transferred WT mice (WT-AM) were sorted as control. RNA was isolated from all input cells, output cells, and WT-AMs, and microarrays were performed.
(A) Schematic overview of the experiment.
(B) PCA of transcriptional profiles of all input cells, output cells, and WT-AMs.
(C) Hexagon and rose diagram of all arrayed genes for YS-Mac-AM, FL-MO-AM, and BM-MO-AM. See Figure S6 and Table S2 for plots and data of the specific modules, individual GO terms, and gene lists.
(D) A list of AM signature genes was published previously (Gautier et al., 2012). Expression of AM signature genes is shown as fold change compared to the reference expression. Genes associated with lipid metabolism (according to Ingenuity Pathway Analysis) are shown in bold.
Data are obtained from four independent replicates from different experiments.
Immunity  , DOI: ( /j.immuni )
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6. Figure 5
YS-Mac-, FL-MO-, and BM-MO-AMs Display Almost Identical Gene Expression
Microarray data were obtained as described for Figure 4.
(A–E) Gene expression profiles of each output cell type were compared to the expression profile of WT-AMs. Differentially expressed genes (DEGs) were identified as at least 2-fold differentially expressed (p < 0.01) in at least one comparison. The 104 unique genes present in the 4 DEG lists were sorted according to at least 1.5-fold differentially expressed by 1, 2, 3, or 4 types of output cells. Schematic overview of the analysis in (A). Fold expression for DEGs up- or downregulated ≥ 1.5-fold by all output cells (B), 3 out of 4 cell types (C), 2 out of 4 cell types (D), or in only one cell type (E). Dotted lines divide groups of genes with similar expression in certain cell types.
(F and G) Gene expression profiles of YS-Mac-AMs, FL-MO-AMs, and BM-MO-AMs were compared in a 1-to-2-comparison, identifying 19 unique genes with at least 2-fold differential expression (p < 0.01). Schematic overview of the analysis in (F). Fold expression of DEGs in (G). The dotted line divides genes differentially expressed in YS-Mac- and BM-MO-AM.
(H and I) Csf2rb−/− pups were transferred with YS-Macs, FL-MOs, and BM-MOs in a 1:1:1 ratio as described for Figure 2. Shown are representative flow cytometry plots (H) and mean ± SEM MHC-II expression (I) by AMs and by recipient CD11c+MHC-II+ dendritic cells (DCs) as control present in the lungs 10 weeks after transfer.
(J) Differential expression of the 22 unique genes identified in (E) and (G) plotted in hexagon diagrams of precursors and AMs.
Data are obtained from four independent replicates from different experiments (A–G, J) and from a representative experiment (four to five mice per time point) of two independent experiments (H and I).
Immunity  , DOI: ( /j.immuni )
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7. Figure 6
Similar Functionality of YS-Mac-, FL-MO-, BM-MO-, and BAL-AM-Derived AMs
Csf2rb−/− pups were transferred with YS-Macs, FL-MOs, BM-MOs, or BAL-AMs and sacrificed 6 weeks or 1 year after transfer. Csf2rb−/− mice transferred with PBS and non-transferred age-matched WT mice served as controls.
(A and B) WT and transfer-derived AMs isolated from 1-year-old mice were allowed to internalize beads coupled to IgG-OVA for 4 hr. Shown are maximum projections of z-stacks (step width: 150 nm) of representative examples of each cell type (A; scale bar represents 10 μm) and mean ± SEM percentage of cells that had phagocytosed at least 1 bead (B).
(C) Splenic CD11b+ conventional DCs or WT-AMs or transfer-derived AMs isolated from 1-year-old mice were co-cultured with OVA protein and CFSE-labeled OT-II T cells. Cultures of T cells and DCs without OVA protein served as controls (no OVA control). Shown are mean ± SEM numbers of proliferated T cells at day 5. Statistical tests compared to DCs.
(D and E) Protein concentrations in the BAL 6 weeks (D) or 1 year (E) after transfer, shown for individual mice and as mean ± SEM per group.
Data were pooled from three (A, B), two (C), at least three (D), and at least two (E) independent transfer-experiments per cell type. 12 controls were derived from experiments that included conditions presented in Figures 6D and 7D and are therefore shown twice. See also Figure S7.
Immunity  , DOI: ( /j.immuni )
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8. Figure 7 Transfer of Tissue Macrophages Does Not Prevent PAP
Csf2rb−/− pups were transferred with peritoneal macrophages (PMacs), Kupffer cells (KCs), or colon macrophages (colon-Mac) and sacrificed 6 weeks after transfer. Csf2rb−/− mice transferred with PBS and non-transferred 6-week-old WT mice served as controls.
(A) Schematic overview of the experiment.
(B) Expression of CD11b, F4/80, CD11c, and SiglecF by CD45.1+ transfer-derived cells.
(C) Numbers of WT and CD45.1+ transfer-derived F4/80hiCD11chiSiglecFhi AMs in the BAL of WT and recipient Csf2rb−/− mice shown per mouse and as mean ± SEM per group.
(D) Protein concentrations in the BAL shown for individual mice and as mean ± SEM per group.
Data were pooled from at least two independent transfer experiments per cell type. 12 controls were derived from experiments that included conditions presented in Figures 3C and 7C and in Figures 6D and 7D and are therefore shown twice.
Immunity  , DOI: ( /j.immuni )
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9. Immunity 2016 44, 755-768DOI: (10.1016/j.immuni.2016.02.017)
Copyright © 2016 Elsevier Inc. Terms and Conditions