1 Supplemental Fig. S1 Supplemental Fig. S1. (A) Diagram of tissue processing used to enrich lung tissue cell populations as described in the Materials and Methods section. (B and C) Homogeneous distribution of immune response cells in normal lung tissue. The anatomical locations of lung tissues examined were based on a silicone rubber cast image of a monkey's lung (1) as shown in Panel B. Flow cytometry analyses of myeloid HLA-DR- and CD11b-positive cells isolated from different lobes of the lung were found to be similar to each other as shown in Panel C.
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Supplemental Fig. S1
Supplemental Fig. S1. (A) Diagram of tissue processing used to enrich lung tissue cell
populations as described in the Materials and Methods section. (B and C) Homogeneous
distribution of immune response cells in normal lung tissue. The anatomical locations of lung
tissues examined were based on a silicone rubber cast image of a monkey's lung (1) as shown
in Panel B. Flow cytometry analyses of myeloid HLA-DR- and CD11b-positive cells isolated
from different lobes of the lung were found to be similar to each other as shown in Panel C.
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Supplemental Fig. S2
Supplemental Fig. S2. Detailed characterization of AM sub-populations. Panel A shows
the gating strategy for sorting AM [AM-H (CD206hiCD163hi) and AM-L
(CD206intCD163int)]. Analysis by size (SSC) and granularity (FSC) also are shown and
followed by Wright-Giemsa staining of each subpopulation. Images were acquired under light
microscopy at 400X magnification. Results shown are representative of three monkeys. Panel
B shows the SSC and FSC comparison between these two AM sub-populations and
Student’s t Test was used to analyze the mean differences.
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Supplemental Fig. S3
Supplemental Fig. S4. In vivo BrdU labeling to assess alveolar macrophages turnover.
Panel A shows the schedule for BrdU/Edu injections (green arrows) of 10 rhesus macaques and
BAL collections from two animals at each time point (blue arrows). Panel B demonstrates that
uptake of BrdU or EdU by AM was low at each time point testing, indicating that these cells
exhibit a low turnover rate.
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Supplemental Table IA. Antibodies used for flow cytometry
Antibody Clone Fluorochrome Vendor
BrdU 3D4 FITC BD
CD1c (BDCA-1) AD5-8E7 Allophycocyanin Miltenyl Biotec
CD11b ICRF44 AL700 BD
CD11c 3.9 Allophycocyanin eBioscience
CD14 RMO52 ECD BD
CD14 M5E2 PB BD
CD16 3G8 V500 BD
CD16 3G8 Allophycocyanin-H7 BD
CD123 7G3 PCP-Cy5.5 BD
CD163 Mac 2-158 PE Trillium
CD192/CCR2 48607 PE R&D
CD195/CCR5 3A9 Allophycocyanin BD
CD197/CCR7 150503 V450 BD
CD20 2H7 eFluor 450 eBioscience
CD206 19.2 Allophycocyanin BD
CD206 15.2 Allophycocyanin-Cy7 Biolegend
CD209 120507 PE R&D
CD3 SP34-2 Pacific Blue BD
CD31 WM59 FITC BD
CD36 FA6-152 Allophycocyanin Beckman Coulter
CD4 L200 PCP-Cy5.5 BD
CD54 LB-2 PE BD
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CD62L MEL-14 PE BD
CD64 10.1 FITC BD
CD68 KP1 FITC DAKO
CD69 TP1.55.3 PE Beckman Coulter
CD71 L01.1 FITC BD
CD8 SK1 V500 BD
CD8 M‐T807 QDot655 NHP Reagent
CD95 DX2 PB ebioscience
HLA-DR L243 (G46-6) PE-Cy7 BD
HLA-DR L243 (G46-6) V450 BD
Mac387 Mac387 AL488 AbD Serotec
TLR1 Gd2.F4 PE ebioscience
TLR2 IMG-416AF488 Al488 Imagenex
TLR9** 26C593.2 PE Imagenex
CD3 SP34-2 V500 BD
CD20 L27 V500-C BD
TNF-α MAb11 Allophycocyanin BD
** Specific fluorochrome-conjugated secondary antibodies applied in the study
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Supplemental Table IB. Summary of phenotype characteristics of blood monocytes, IM,
and AM by flow cytometry
Antigen
Description
CD14+
monocyte
IM
AM
CD11b Adhesion molecule, myeloid cells marker + + low
CD11c Adhesion molecule, DC marker low/p+* Low/p+ +
CD14 LPS activation cascade, monocyte marker + + -
CD16 Fc receptor, monocyte activation marker - /p+ p+ -
CD163 Haptoglobin-hemoglobin (Hp-Hb)
scavenger receptor, macrophage marker
+ + +
CD192/CCR2 Chemokine receptor, HIV coreceptor,
monocyte migration
+ - -
CD195/CCR5 Chemokine receptor, HIV coreceptor - - -
Cd197/CCR7 Chemokine receptor/ cell migration - - -
CD1c (BDCA-1) DC marker - - -
CD206 Mannose receptor, macrophage marker - - +
CD209 C-type lectin DC-SIGN low low low
CD31 Platelet endothelial cell adhesion
molecule 1 (PECAM-1)
+ + +
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CD36 Class B scavenger receptor for oxidized
lipids (oxLDL), thrombospondin-1 and
collagen type I
low &
high
high high
CD4 HIV core receptor - - -
CD54 Adhesion molecule, activation marker for
antigen presenting cell
- - -
CD62L L-selectin, cell adhesion molecule - - -
CD64 Fc receptor for immunoglobulin IgG + + +
CD68 Lysosome marker, monocyte macrophage
marker
+ + +
CD69 Activation marker - - -
CD71 Transferrin receptor, macrophage
activation
- - -
CD95 Fas ligand, induce apoptosis + + +
HLA-DR MHCII, antigen presenting cell marker, T
cell activation marker
+ + high
Mac387 Monocyte, macrophage marker, also
stains granulocyte
+ + -
TLR1 PRR, recognize lipoproteins - -/low -
TLR2 PRR, recognize peptidoglycan (PGN) low low low
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TLR9 PRR, recognizes unmethylated CpG
oligonucleotide (ODN)
+ + +
* p+ ; partial positive
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References Cited
1. Carson, J. P., D. R. Einstein, K. R. Minard, M. V. Fanucchi, C. D. Wallis, and R. A. Corley. 2010. High resolution lung airway cast segmentation with proper topology suitable for computational fluid dynamic simulations. Computerized medical imaging and graphics : the official journal of the Computerized Medical Imaging Society 34: 572-578.
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