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1 SUPPLEMENTARY INFORMATION doi: /nature19814 Figure 3e Beads: Hep SA Sup Hep SA Sup Hep - SA - Sup Blot: NP-1 Blot: MECA-32 Blot: VEGF Figure 3f Rbt IgG ctrl IP VEGF IP Extended Data Figure 9d Beads: CD4-Fc SA Hep SA Hep Loading PLVAP Blot: MECA M NaCl 50 Rbt IgG ctrl IP VEGF IP VEGF (dimer; non-reducing gel) Beads: CD4-Fc SA Hep SA Hep Loading Blot:anti-human IgG Blot: VEGF Figure 3g Beads: SA Hep CD4-Fc SA Hep Loading Extended Data Figure 9e Beads: Hep Hep Hep SA Blot:anti-human IgG Competition - FN ColI - Blot:anti-human IgG Supplementary Figure 1. The gel source data. The uncropped original blots from electrophoretic experiments. Shown in Fig.3e, 3f, 3g, Extended Data Fig. 9d and 9e 1
2 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 1: Full flow cytometric gating strategy of progenitor and myeloid leukocytes Adults Tissue leukocyte type Full gating used (a,b,c,d,e) Additional markers used References Spleen Embryonic-derived tissue resident macrophages Live/CD45 neg /CD8 neg /CD4 CD206 + / CSF-1R Bone marrow-derived macrophages Live/CD45 neg /CD8 neg /CD4 /F4/80 Int CSF-1R + 7,11,20 Recently entered tissue monocytes Live/CD45 neg /CD8 neg /CD4 neg / F4/80 Int /CD11b /Ly6c Low or hi CSF-1R + 20,50 Peritoneal macrophages Embryonic-derived tissue resident macrophages Live/CD45 neg /CD8 neg /CD4 /F4/80 CSF-1R + MHC II 7,11,20,25 low/neg 11,20,51-54 Bone marrow -derived macrophages Lung Live/CD45 neg /CD8 neg /CD4 Int CSF-1R + MHC II + 11,20,51,52 Embryonic-derived alveolar macrophages Live/CD45 neg/ CD8 neg /CD4 + /CD11c F4/80 high /CD206 + /SiclecF high /CSF-1R +7,11,18,20 Blood Bone marrow-derived infiltrating monocytes Live/CD45 + /Ly6C 26,55 Bone marrow-derived patrolling monocytes Live/CD45 + /Ly6C Peripheral lymph node Low 26,55 Bone marrow-derived macrophages Live/CD45 + /CD11b Int Colon Bone marrow-derived macrophages Live/CD45 + /CD11b Int 23,56 Liver Embryonic-derived tissue resident macrophages Live/CD45 neg /CD8 neg /CD4 Bone marrow Low or hi /Ly6c CSF-1R + 7,11,20,57 Bone marrow-derived macrophages Live/CD45 neg /CD8 neg /CD4 /F4/80 Int CSF-1R + 7,11,20,57 Recently entered tissue monocytes Live/CD45 neg /CD8 neg /CD4 neg / F4/80 Int /CD11b 20 Hematopoietic stem cells (HSC) Live/Lin neg /C-kit + /Sca Common myeloid progenitor (CMP) Live/Lin neg /C-kit + /Sca-1 Neg Neg /IL7R 58 Common lymphoid progenitor (CCP) Live/Lin neg /C-kit + /Sca-1 Low /IL7R + 58 Ly6C high monocytes Live/CD45 + /CD11b + /Ly6C 59 Ly6C low monocytes Live/CD45 + /CD11b + Low /Ly6C 59 Brain Embryonic-derived microglia cells Live/CD45 Low / F4/80 6,7,20,60 Mammary gland Macrophages Live/CD45 neg /CD
3 SUPPLEMENTARY INFORMATION RESEARCH Embryos Tissue leukocyte type Full gating used Additional markers used References Spleen Yolk sac-derived embryonic macrophages Live/CD45 CSF-1R + 7,11,20,25 Fetal liver-derived embryonic macrophages Live/CD45 Int CSF-1R + 7,11,20,25 Lung Yolk sac-derived embryonic macrophages Live/CD45 CSF-1R + 7,11,20,61 Fetal liver-derived embryonic macrophages Live/CD45 Int CSF-1R + 7,11,20 Blood Blood monocytes Live/CD45 + /Ly6C 26,55 Yolk sac-derived embryonic macrophages Live/CD45 Ly6C neg /CSF-1R + 7,11,20 Fetal liver-derived embryonic macrophages Live/CD45 /F4/80 Int Ly6C + /CSF-1R + 7,11,20 Macrophage-dendritic cell progenitor (MDP) Live/CD45 + /CD11b neg /CSF-1R + /c-kit + /Flt-3 + Neg /Ly6C 17,62 Fetal liver myeloid precursor (MP) Live/CD45 + /CD11b neg /CSF-1R + /c-kit + /Flt-3 Neg Neg /Ly6C 17,62 Common monocyte precursor (cmop) Live/CD45 + /CD11b neg /CSF-1R + /c-kit + /Flt-3 Neg /Ly6C + 17,62 Fetal liver Ly6C + monocytes Live/CD45 + /CD11b + /CSF-1R + /c-kit neg /Flt-3 Neg /Ly6C + 17,62 Fetal liver Ly6C neg monocytes Live/CD45 + /CD11b + /CSF-1R + /c-kit neg /Flt-3 Neg Neg /Ly6C 17,62 Erythro-myeloid progenitor (EMP) Live/CD41 + /CD45 + /c-kit Neg /F4/80 10,17 Hematopoietic stem cells (HSC) Live/c-kit + /Sca ,17,63 Brain Embryonic-derived microglia cells Live/CD45 Low / F4/80 6,7,16,20,60 Yolk sac Yolk sac embryonic macrophages Live/CD45 + /CD11b CSF-1R + 7,8,11,20 Yolk sac embryonic macrophages (MAC) Live/CD41 + /CD45 /c-kit Neg CSF-1R + 10,21 Erythro-myeloid progenitor (EMP) Live/CD41 + /CD45 + /c-kit /F4/80 Neg CSF-1R + 10,17 a) The selected markers by which the cell types are referred to in the text are shown in bold. b) Live = 7-Amino-Actinomycin (7-AAD) negative c) Int = intermediate, neg = negative, d) Lin = Linage marker (CD3e, CD11b, B220, TER-119, Ly6G/Ly6C; , BD) e) Lymphocytes from different adult tissues were all gated as follows: B lymphocytes Live/CD45 + /CD8 neg /CD4 neg /B220 + CD4 T-helper lymphocytes Live/CD45 neg /CD8 neg /CD4 + CD8 T-cytotoxic lymphocytes Live/CD45 neg /CD4 neg /CD Swirski, F. K. et al. Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science 325, (2009). 51. Rosas, M. et al. The transcription factor Gata6 links tissue macrophage phenotype and proliferative renewal. Science 344, (2014). 52. Nguyen, H. H., Tran, B. T., Muller, W. & Jack, R. S. IL-10 acts as a developmental switch guiding monocyte differentiation to macrophages during a murine peritoneal infection. J. Immunol. 189, (2012). 53. Gautier, E. L. et al. Gata6 regulates aspartoacylase expression in resident peritoneal 3
4 RESEARCH SUPPLEMENTARY INFORMATION 53. Gautier, E. L. et al. Gata6 regulates aspartoacylase expression in resident peritoneal macrophages and controls their survival. J. Exp. Med. 211, (2014). 54. Ghosn, E. E. et al. Two physically, functionally, and developmentally distinct peritoneal macrophage subsets. Proc. Natl Acad. Sci. USA 107, (2010). 55. Geissmann, F., Jung, S. & Littman, D. R. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 19, (2003). 56. Bain, C. C. et al. Resident and pro-inflammatory macrophages in the colon represent alternative context-dependent fates of the same Ly6Chi monocyte precursors. Mucosal Immunol. 6, (2013). 57. Movita, D. et al. Kupffer cells express a unique combination of phenotypic and functional characteristics compared with splenic and peritoneal macrophages. J. Leukoc. Biol. 92, (2012). 58. Adolfsson, J. et al. Identification of Flt3+ lympho-myeloid stem cells lacking erythromegakaryocytic potential a revised road map for adult blood lineage commitment. Cell 121, (2005). 59. Hanna, R. N. et al. The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C- monocytes. Nat. Immunol. 12, (2011). 60. Gomez Perdiguero, E., Schulz, C. & Geissmann, F. Development and homeostasis of resident myeloid cells: the case of the microglia. Glia 61, (2013). 61. Zaynagetdinov, R. et al. Identification of myeloid cell subsets in murine lungs using flow cytometry. Am. J. Respir. Cell Mol. Biol. 49, (2013). 62. Hettinger, J. et al. Origin of monocytes and macrophages in a committed progenitor. Nat. Immunol. 14, (2013). 63. Mebius, R. E. et al. The fetal liver counterpart of adult common lymphoid progenitors gives rise to all lymphoid lineages, CD45+CD4+CD3 cells, as well as macrophages. J. Immunol. 166, (2001). 4
5 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Table 2: Antibodies used in flow cytometry and immunohistochemistry Flow cytometry Antigen Antibody clone Fluorochrome a) Catalog # Company b) B220 RA3-6B2 PE BD B220 RA3-6B2 BV BD B220 RA3-6B2 Pacific Blue BD CD4 GK1.5 APC-Cy BD CD4 RM4-5 A BD CD4 RM4-5 PE BD CD PerCP-Cy5, BD CD PE BD CD BV BD CD11b M1/70 APC-Cy BD CD11b M1/70 BB BD CD11b M1/70 PE BD CD11c HL3 BV BD CD11c HL3 PerCP-Cy5, BD CD34 HM34 PerCP-Cy5, Biolegend CD41 MWReg30 PE BD CD41 MWReg30 FITC BD CD45 30-F11 APC-Cy BD CD45 30-F11 PerCP-Cy5, BD CD45 30-F11 V BD CD115 (CSF-1R) AFS98 Pe-Cy ebiosciences CD117 (C-kit) ACK2 BV Biolegend CD127 (IL-7) SB/199 Pe-Cy BD CD135 (Flt-3) A2F10.1 BV BD CD206 MR5D3 A488 MCA2235A488 Abd Serotec CD304 3DS304M PE ebiosciences F4/80 Cl:A3-1 A647 MCA497A647 Abd Serotec F4/80 Cl:A3-1 PE Ab Abcam Lin 145-2C11, M1/70, RA3-6B2, APC BD TER-119, RB3-8C5 Ly6C AL-21 BV BD Ly6C AL-21 FITC BD Ly6G 1A8 BV Biolegend MHCII M5/ Pe-Cy ebiosciences Sca PE FAB1226P R&D Systems Siglec F E PE-CF BD VEGFR PE FAB4711P R&D Systems VEGFR2 Avas12a1 PE ebiosciences Isotype controls Armenian Hamster IgG1, λ1 PerCP-Cy5, BD Armenian Hamster IgG1, λ2 BV BD Armenian Hamster IgG PerCP-Cy5, Biolegend 5
6 RESEARCH SUPPLEMENTARY INFORMATION Rat IgG1 FITC BD Rat IgG2a A488 MCA1212A488 Abd Serotec Rat IgG2a A BD Rat IgG2a BV BD Rat IgG2a BV BD Rat IgG2a BV BD Rat IgG2a Pacific Blue BD Rat IgG2a PE-CF BD Rat IgG2a PerCP-Cy5, BD Rat IgG2a PE-Cy ebiosciences Rat IgG2b APC-Cy BD Rat IgG2b A Biolegend Rat IgG2b BB BD Rat IgG2b PerCP-Cy5, BD Rat IgG2b V BD Rat IgG2b BV Biolegend Rat IgG2b V BD Rat IgG2b BV Biolegend Rat IgG2b PE-Cy ebiosciences Rat IgM FITC BD Lineage Isotype Control Cocktail APC BD Immunohistochemistry Antigen Antibody clone Fluorochrome Catalog # Source Caveolin Rabbit polyclonal unconjucated sc-894 Santa-Cruz CD31 MEC13.3 APC BioLegend CD117 2B8, Rat IgG2b unconjucated BD F4/80 BM8 A488 MF48020 Invitrogen Lyve-1 Rabbit polyclonal unconjucated 102-PA50AG/103- Reliatech PA50 MAdCAM-1 MECA-367, Rat IgG2a unconjucated E.Butcher Stanford Univ. Plvap Meca-32, Rat IgG2a unconjucated MECA AK Invivo 2028/01 Plvap Meca-32, Rat IgG2a unconjucated BD Plvap PAL-E unconjucated ab8086 Abcam VEGF Rabbit polyclonal unconjucated Ab46154 Abcam Second stage antibodies Donkey anti Rat IgG A488 A21208 Life Technologies Goat anti Mouse IgG A488 A11029 Invitrogen Goat anti Rabbit IgG A546 A11035 Invitrogen Goat anti Rat IgG A546 A11081 Life Technologies Goat anti Rat IgG A647 A21247 Life Technologies Goat anti Rabbit IgG A633 A21071 Invitrogen a) APC, Allophycocyanin; APC-Cy7, Allophycocyanin-indotricarbocyanine; A488, Alexa Flour 488; A647, Alexa Fluor 647; BB515, Brilliant Blue 515; BV421,Brilliant Violet 421; BV510, Brilliant Violet 510; BV650, Brilliant Violet 650; FITC, Fluorescein isothiocyanate; PE, Phycoerythrin; Pe-Cy7,Phycoerythrin-indotricarbocyanine; PerCP-Cy5,5, Peridinin chlorophyll protein cyanine 5.5 b) BD, Becton-Dickinson 6
7 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Table 3: Cell numbers in WT and Plvap / mice (a) Embryos WT Plvap / E10.5 Yolk-sac Total leukocytes (x10 6 ) 0.16 ± ± E12.5 Liver Total leukocytes (x10 6 ) 2.84 ± ± 1.54 HSC 3024 ± ± Yolk-sac Total leukocytes (x10 6 ) 2.81 ± ± E14.5 Liver Total leukocytes (x10 6 ) ± ± 2.30 CD11b F4/80 Int 3388 ± ± 11 b CD11b + F4/ ± ± 1419 Lung Total leukocytes (x10 6 ) 1.22 ± ± 0.19 b E16.5 Liver Total leukocytes (x10 6 ) ± ± 9.83 CD11b + F4/80 Int 3970 ± ± CD11b F4/ ± ± Blood CD11b + Ly6C ± ± b Lung CD11b + F4/80 Int 5838 ± ± CD11b F4/ ± ± Spleen CD11b + F4/80 Int 8339 ± ± b CD11b F4/ ± ± 1385 Adults Blood (Cells x 10 3 / ml of blood) WT Plvap / White blood cells 8.33 ± ± 1.80 Lymphocytes 5.57 ± ± 1.23 Monocytes 0.17 ± ± 0.11 Neutrophils 2.56 ± ±
8 RESEARCH SUPPLEMENTARY INFORMATION Spleen Weight (g) ± ± Total leukocytes after red blood lysis (x10 6 ) ± ± CD11b F4/80 Int 2972 ± ± CD11b + F4/ ± ± b B cells ± ± 3299 Liver Weight (g) 1.47 ± ± 0.27 CD11b F4/80 Int 1783 ± ± CD11b + F4/ ± ± b B cells 4435 ± ± Lung CD11b + CD11c 1163 ± ± b Peritoneal cells CD11b F4/ X 10 6 ± ± b Brain CD45 low F4/80 HI ± ± 6906 PLN Total leukocytes (x10 6 ) 4.78 ± ± 1.26 B cells 8319 ± ± 1537 b Bone marrow Total leukocytes (x10 6 ) ± ± 1.82 Mammary gland CD11b + F4/ ± ± b a) Cell numbers are shown as mean±sem (n=3-5/mice/genotype), determined from equal-sized tissue pieces or equal volumes of blood (the values can be compared between the two genotypes on each row, but not between the different rows). not between the diff b) p<0,05 8
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