1. Volume 46, Issue 3, Pages 504-515 (March 2017)
Dendritic Cells Display Subset and Tissue-Specific Maturation Dynamics over Human Life 
Tomer Granot, Takashi Senda, Dustin J. Carpenter, Nobuhide Matsuoka, Joshua Weiner, Claire L. Gordon, Michelle Miron, Brahma V. Kumar, Adam Griesemer, Siu-Hong Ho, Harvey Lerner, Joseph J.C. Thome, Thomas Connors, Boris Reizis, Donna L. Farber 
Immunity 
Volume 46, Issue 3, Pages (March 2017)
DOI: /j.immuni
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2. Immunity 2017 46, 504-515DOI: (10.1016/j.immuni.2017.02.019)
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1 Identification of cDC Subsets in Diverse Human Tissue Sites
(A) Schematic showing each of the 78 donors used in this study is designated by an individual symbol with the following characteristics indicated: Gender (filled symbol, male; open symbol, female), race (green, Caucasian/White; red, African American/Black; blue, Hispanic; and orange, Asian), and cause of death (upward triangle, head trauma; downward triangle, stroke; circle, anoxia). Asterisk indicates male donor for whom no other information was available.
(B) Left: gating strategy for the identification of cDC1 and cDC2 in multiple tissues obtained from human organ donors. Cells were initially gated on live (DAPIlo) CD45+ singlets (not shown), then on CD11c+HLA-DR+CD14−, lineage (Lin)-neg (Lin = CD3, CD15, CD19, CD20, CD56) and CD1c+ or CD141+ to identify cDC populations and further subdivided by CD141+CD13+ and CD1c+ to delineate cDC1 and cDC2, respectively. Right: expression of key cDC markers CD141, CD13, Sirp-α, and CD1c by cDC1 (red) and cDC2 (blue) subsets. Data derive from Donor 233.
(C) Plots show expression of different markers (y axis) on cDC1 (red), cDC2 (blue), and CD141+ CD1c+ (tan) cells.
(D) Histograms show expression of CD26 on the subsets designated in (C).
(E) Compiled Clec9a, CD26, and Sirp-α expression by cDC1 and cDC2 delineated as in (B), expressed as geometric mean fluorescence intensity (gMFI) ± SEM from 7–29 donors for each tissue. Tissue abbreviations: BM = bone marrow; TLN = tracheal LN; LLN = lung LN; PLN = pancreatic LN; MLN = mesenteric LN; ILN = iliac LN.
(F) Fluorescence staining of LLN sections from a single donor showing nuclear IRF8 staining on CD13+Clec9A+ cells displaying a DC morphology in the T cell zone (green) (upper image) and nuclear IRF4 staining by CD1c+ cells with a DC morphology (lower right). Shown in lower left is control staining for CD1c expression on CD19+ B cells (blue), which lack IRF4 expression. Images are shown at 200× magnification. Scale bar, 20μm.
Immunity  , DOI: ( /j.immuni )
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4. Figure 2
Tissue Distribution of Monocytic Cells and DC Subsets in the Human Body
(A) Compiled frequencies (mean ± SEM) of cDC1, cDC2, pDCs, and CD14+ cells gated as in Figures 1 and S1, expressed as %CD45+ cells, compiled from 10–50 donors for each tissue site. Secondary lymphoid tissues are shaded in purple, and mucosal tissues are shaded in tan; blood-rich sites are highlighted in gray. Tissue abbreviations are designated in Figure 1E, and Bld = blood; Spl = spleen; Lng = lung; Jej = jejunum; Ile = ileum; Col = colon; PP = Peyers Patches; App = appendix.
(B) Compiled pie charts depict the ratios (mean from each site) of DC subsets and monocytes analyzed in 14 different tissues, with the perimeter of the circle denoting each tissue grouping: blood-rich tissues (gray arc), secondary lymphoid tissues (purple arc), and mucosal tissues (tan arc). The proportions of each subset are denoted by different colors: CD14+ monocytes (green), pDCs (orange), and cDCs (dark blue), which are further subdivided into cDC2 (light blue) and cDC1 (red).
(C) Graphical representation of cDC distribution plotted as cDC1 frequency (%CD45+ cells, y axis) versus cDC2:cDC1 ratio (x axis) for each tissue. Five tissue groupings are delineated by shaded circles into circulatory, lymphoid tissue (subdivided into peripheral lymphoid sites and gut-associated lymphoid tissues [GALT, appendix and Peyer’s Patch]), mucosal tissues, and lung and lung-draining LN (see text).
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5. Figure 3
Subset and Regional Differences in cDC Maturation in Secondary Lymphoid Organs
(A) HLA-DR expression (gMFI ± SEM) on cDC1 and cDC2 from 14 different tissues compiled from 10–30 donors per tissue.
(B) Top: differential HLA-DR expression by LN cDCs delineating immature (HLA-DRlo) and mature (HLA-DRhi) populations. Bottom: cDC subset delineation of immature versus mature populations. Representative data from donor 223.
(C) Ratio of mature to immature cDCs in spleen and five different LNs with TLN (tracheal LN), LLN (lung LN), PLN (pancreatic LN), MLN (mesenteric LN), and ILN (iliac LN). Significance was determined using paired t tests.
(D) cDC2:cDC1 ratio of immature versus mature cDCs in LNs shown in (C).
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <
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6. Figure 4
Differential Clustering and Density of cDC Subsets in Lung-Draining and Mesenteric LNs
(A) Visualization of cDC subsets in human LNs from a single representative donor (donor 250). Fixed human LLN and MLN were stained for CD3 (blue), HLA-DR (green), Clec9A (red), and CD1c (white). Images a and b show whole LN at 10× magnification, with the boxed regions shown below in images c–f at 20× magnification. Green arrows indicate B cell follicles (c and d); red arrows indicate cDC1, and white arrows indicate cDC2 (e and f). Images are representative of at least five different donors. Scale bars, a and b, 1000μm; c and d, 200μm; e and f, 100μm.
(B) Quantification of cDC2 density in T cell zones of LLN and MLN obtained from 17 donors calculated by dividing the number of cDC2s (using Imaris software) by the T cell zone area, defined using ImageJ software (see Experimental Procedures).
∗p < 0.05, as calculated by a paired t test.
Immunity  , DOI: ( /j.immuni )
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7. Figure 5
LN Mature cDCs Display Characteristics of Tissue Migratory Cells
(A) HLA-DR and CCR7 expression by lung (left) and LLN (right) cDC1 and cDC2 of two donors (233, upper, and 223, lower).
(B) Graph shows the ratio of mature:immature cDC2s in the LLN plotted versus the ratio of mature:immature cDCs in the lung compiled from 20 donors. Shaded area (gray) delineates region of greater maturation in the LLN compared to the lung.
(C) Graphs show ratios of cDC2:cDC1 in the lung (left) and LLN (right) as a function of LLN cDC2 mature:immature ratios compiled from 20 (left) and 53 (right) donors.
(D) CD103 and CD1a expression by cDC2 in the jejunum (light blue) and lung (dark blue) shown in representative flow cytometry plots (left) and compiled gMFI from 5–16 donors (right).
(E) CD103 and CD1a expression by HLA-DRlo and HLA-DRhi cDC2 in three different LNs shown in representative flow cytometry plots (left) from individual donors (donor 215, top; donor 297, bottom) and compiled results calculating the ratio of CD103+:CD103− (top) or CD1a+:CD1a− (bottom) cDC2 from 4–22 donors (right). Lung sections were taken from the lateral basal region for each donor.
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <
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8. Figure 6 cDC Subset Frequency in Tissues over the Human Lifespan
(A) Graphs show compiled frequencies of cDC1 (left) and cDC2 (right) subsets (% CD45+ cells) in blood and seven different tissues as a function of age. Line shows linear regression.
(B) Representative flow cytometry plots showing cDC1 and cDC2 in the jejunum and MLN of two pediatric and one young adult donor as indicated.
(C) Compiled frequencies of cDC1, cDC2, and CD14+ cells from jejunal and MLN samples of 7–8 children and 25–31 adults.
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9. Figure 7
cDC2 Maturation Dominance Emerges at Infancy and Is Retained over Life
(A) Top: representative flow cytometry plots show frequency of mature (HLA-DRhi) and immature (HLA-DRlo) CD11c+ cDCs in LLN and MLN of individual donors from 3 months to 93 years of age. Bottom: graphs show ratio of mature:immature cDCs in LNs plotted as a function of age, with line indicating linear regression.
(B) Top: representative flow cytometry plots show frequency of cDC1 and cDC2 in the mature and immature cDC fraction in the LLN and MLN of individual donors of indicated ages. Bottom: ratio of cDC2:cDC1 in the mature and immature compartments of different LNs plotted as a function of age with line indicating linear regression.
∗p < 0.05.
Immunity  , DOI: ( /j.immuni )
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