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2.
Figure 4

Figure 4. From: Human Tissues Contain CD141hi Cross-Presenting Dendritic Cells with Functional Homology to Mouse CD103+ Nonlymphoid Dendritic Cells.

CD141hi DCs Migrate to Skin-Draining Lymph Node
(A) Identification of migratory (mig) and resident (res) DCs in mechanically dispersed dermatopathic LN (left) and tonsil (right).
(B) Comparison of CD141hi and CD1c+ DCs in migratory (mig) and resident (res) fractions. Relative expression of CD1c, CD1a, and langerin by CD1c+ DCs (green), CD141hi DCs (red), and cells from epidermal LC gate (purple).
(C) Relative expression of CD80, CD83, CD86, and CCR7 by migratory (tinted) and resident CD14+ DCs (blue), CD1c+ DCs (green), and CD141hi DCs (red) compared to isotype (gray).
Representative data from four dermatopathic LNs and four tonsils are shown.

Muzlifah Haniffa, et al. Immunity. 2012 July 27;37(1):60-73.
3.
Figure 6

Figure 6. From: Human Tissues Contain CD141hi Cross-Presenting Dendritic Cells with Functional Homology to Mouse CD103+ Nonlymphoid Dendritic Cells.

CD141hi DCs Synthesize CXCL10 and TNF-α but Not IL-12
(A) IL-12p70 and IL-23p19 production from unstimulated (−), LPS (LPS)-, poly(I:C) (pIC)-, a cocktail of TNF-α, IL-1β, IFN-α, IFN-γ, LPS, and pIC (c)-, and cocktail with the addition of GM-CSF (c + GM-CSF)-stimulated CD141hi DCs, CD1c+ DCs, CD14+ DCs, and LCs with mo-DCs as control. Composite results from six donors are shown, mean ± SEM. Asterisk denotes subset not analyzed.
(B) CXCL10, TNF-α, IL-1β, IL-6, IL-8, IL-10, IL-23p19, and IL-12p70 production from CD141hi DCs, CD1c+ DCs, CD14+ DCs, and LCs as stimulated in (A). Composite results from four donors are shown, mean ± SEM. Asterisk denotes subset not analyzed.

Muzlifah Haniffa, et al. Immunity. 2012 July 27;37(1):60-73.
4.
Figure 1

Figure 1. From: Human Tissues Contain CD141hi Cross-Presenting Dendritic Cells with Functional Homology to Mouse CD103+ Nonlymphoid Dendritic Cells.

Identification of CD141hi DCs in Human Tissues
(A) Flow cytometry of peripheral blood, collagenase-treated whole skin, liver, and mechanically dispersed lung. Gating strategy used to identify three myeloid DC subsets within LinHLA-DR+ fraction (yellow gate) in tissues: (1) CD14+ DCs (blue gate), (2) CD14CD11c+ DCs (green gate), and (3) CD14CD11cloCD141hi DCs (red gate). Langerinhi epidermal LCs (purple gate) are identifiable in the skin. Representative data from 20 blood, 18 skin, 12 liver, and 8 lung donors are shown.
(B) Relative expression of CD1c, CD1a, and langerin by CD11c+ DCs (green) and CD141hi DCs (red). Representative data from four blood, skin, liver, and lung donors are shown.
(C) Identical gating strategy as (A) to correlate CD141hi cells with established populations of skin DCs from digested dermis and spontaneously migrating DCs from skin explants cultured for 60 hr. Representative data from seven skin donors are shown.
(D) Frequency of CD141hi cells as a percent of CD45+ mononuclear cells in skin, liver, and lung relative to peripheral blood. Composite data from six blood, skin, liver, and lung donors are shown. p < 0.05, Mann-Whitney U test.
See also Figure S1.

Muzlifah Haniffa, et al. Immunity. 2012 July 27;37(1):60-73.
5.
Figure 3

Figure 3. From: Human Tissues Contain CD141hi Cross-Presenting Dendritic Cells with Functional Homology to Mouse CD103+ Nonlymphoid Dendritic Cells.

Skin CD141hi DCs Potentially Develop from Blood CD141+ DCs
(A and B) Relative expression of (A) CD80, CD83, CD86, and PD-L1 and of (B) CLA and CCR7 by blood and skin CD14+ DCs (blue), CD1c+ DCs (green), and CD141hi DCs (red) compared to isotype (gray). Representative data from three blood, skin, and lung donors are shown.
(C) Relative expression of CD80, CD83, CD86, and CCR7 (blue) to isotype control (gray) by CD1a+CD1c+ and CD1aCD1c fractions of CD141hi cells in the skin (shown with their relative percentages). Representative data from three skin donors are shown.
(D) CD1c and CD1a expression by FACS-purified, Qtracker605-labeled blood CD141+ DCs cultured in medium (−Dermis) or with digesting dermis (+Dermis) and migrated CD141hi cells from 60 hr skin explants cultured ex vivo (Migrants). Representative data from two blood and skin donors for “spiking” experiment and five donors for skin explant migration are shown.
(E) DNA content of FACS-sorted blood and skin CD14+, CD1c+, and CD141hi DCs. Right panel shows percent of DCs in S, G2, or M phase in blood and skin. Representative and composite data from four blood and five skin donors are shown, mean ± SEM. p < 0.05, Mann-Whitney U test comparing skin CD141hi DCs with all other subsets.

Muzlifah Haniffa, et al. Immunity. 2012 July 27;37(1):60-73.
6.
Figure 7

Figure 7. From: Human Tissues Contain CD141hi Cross-Presenting Dendritic Cells with Functional Homology to Mouse CD103+ Nonlymphoid Dendritic Cells.

Transcriptome Mapping of Human and Mouse Nonlymphoid Tissue DCs
Microarray expression profiles were obtained from FACS-purified monocyte and DC subsets from human blood and skin and mouse bone marrow, blood, spleen, liver, and lung.
(A) Cluster dendrogram of human DC and monocyte subsets after removal of tissue-specific genes demonstrating the relationships between blood CD141+ DCs and skin CD141hi DCs (red), blood and skin CD1c+ DCs (green), CD14+ monocytes and DCs (blue), blood CD16+ monocytes (light blue), and pDCs (brown). Microarray profiles were obtained from six blood and four skin donors.
(B) CMAP enrichment scores for skin CD141hi DCs against all human skin and blood monocyte and DC subsets. Scatterplot and mean, a 1,000 permutation test between gene signatures was performed on each enrichment score to determine significance. Enrichment scores for human skin CD141hi DCs with all other human monocyte and DC subsets were significant at p < 0.0001.
(C) CMAP enrichment scores for human (1) CD141 lineage, (2) CD1c lineage, (3) skin CD14+ DCs, and (4) blood CD14+ monocytes against mouse monocyte and DC subsets. CMAP analysis was performed with mouse orthologs of human transcripts. Microarray profiles were obtained from 3–4 experimental sets from 10–15 WT mice per set. Scatterplot and mean, a 1,000 permutation test between gene signatures was performed on each enrichment score to determine significance. Enrichment scores were significant at p < 0.05 for all data sets except for the inverse association between human CD1c lineage with mouse Gr1hi and Gr1lo monocytes.

Muzlifah Haniffa, et al. Immunity. 2012 July 27;37(1):60-73.
7.
Figure 5

Figure 5. From: Human Tissues Contain CD141hi Cross-Presenting Dendritic Cells with Functional Homology to Mouse CD103+ Nonlymphoid Dendritic Cells.

CD141hi DCs Are Superior at Cross-Presenting Soluble Antigen
(A and B) IFN-γ production assessed by ELISpot assay upon cross-presentation of soluble HBsAg by blood and skin DCs to HLA-A0201-restricted s183-91 CD8+ T cell clones at a DC:T cell ratio of 1:2.5. Composite data from four blood and seven skin donors are shown with mean ± SEM. p < 0.05, Mann-Whitney U test, comparing respective experimental condition for CD141+ blood and CD141hi skin DCs with CD1c+ DCs, CD14+ blood monocytes, skin DCs, and LCs.
(C) Uptake of PE-labeled HBsAg and FITC-conjugated Dextran by skin CD141hi DCs (red), CD1c+ DCs (green), CD14+ DCs (blue), and LCs (purple). Representative data from two skin donors are shown; median fluorescent intensity of antigen uptake for each DC subset at 37°C (color) compared to 4°C (gray) are stated in the histogram box.
(D) Alloactivation of CD4+ and CD8+ T cells by skin CD141hi DCs (red), CD1c+ DCs (green), and CD14+ DCs (blue). Proliferation was measured over 6 days by CFSE dilution. Representative dot plots and composite results from four skin donors are shown, mean ± SEM. p < 0.05, ANOVA with post-test Bonferroni correction.

Muzlifah Haniffa, et al. Immunity. 2012 July 27;37(1):60-73.
8.
Figure 2

Figure 2. From: Human Tissues Contain CD141hi Cross-Presenting Dendritic Cells with Functional Homology to Mouse CD103+ Nonlymphoid Dendritic Cells.

CD141hi Tissue DCs Express Markers of Cross-Presenting DCs
(A) LinHLA-DR+ cells from blood and skin were FACS purified according to gating shown in dot plots.
(B) RNA from FACS-purified cells in (A) was analyzed for the expression of XCR1, TLR3, CLEC9A, and CADM1 by qRT-PCR. Data shown are from two blood and skin donors.
(C) DC migration from skin explants cultured ex vivo over 24 hr in medium alone (−) or with XCL1 or CCL3. Composite data from four skin donors are shown, mean ± SEM. p < 0.05, Mann-Whitney U test.
(D) Relative expression of FLT3, CLEC9A, MCSFR, and CX3CR1 by CD14+ DCs (blue), CD1c+ DCs (green), and CD141hi DCs (red) compared to isotype (gray) from blood, skin, and lung. Representative data from three blood, skin, and lung donors are shown.
(E and F) Morphology of FACS-sorted blood and skin CD141+, CD1c+, and CD14+ DCs and monocytes (E) visualized by GIEMSA staining of cytospin preparations (X100) and (F) by SEM. Scale bars represent 10 μm in (E) and 1 μm in (F).
(G) Pseudo-color (×63) images of whole-mount skin immunostained for MHC class II (HLA-DR, HLA-DQ, and HLA-DP) (green), CD11c (blue), and XCR1 (red). White arrows in overlay image (right bottom) highlight the XCR1+CD11cloHLA-DR+ cells corresponding to skin CD141hi DCs. Scale bars represent 5 μm.

Muzlifah Haniffa, et al. Immunity. 2012 July 27;37(1):60-73.

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