Identification of Lineage Relationships and Novel Markers of Blood and Skin Human Dendritic Cells

of December 3, 2012.This information is current as

Dendritic CellsNovel Markers of Blood and Skin Human Identification of Lineage Relationships and

CameronSharon R. Lewin, Anthony L. Cunningham and Paul U.Sandgren, Min Kim, Sarah K. Mercier, Rachel A. Botting, Andrew N. Harman, Chris R. Bye, Najla Nasr, Kerrie J.

ol.1200779http://www.jimmunol.org/content/early/2012/11/25/jimmun

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The Journal of Immunology

Identification of Lineage Relationships and Novel Markers ofBlood and Skin Human Dendritic Cells

Andrew N. Harman,*,1 Chris R. Bye,†,1 Najla Nasr,* Kerrie J. Sandgren,*

Min Kim,* Sarah K. Mercier,* Rachel A. Botting,* Sharon R. Lewin,‡,x,{,||

Anthony L. Cunningham,* and Paul U. Cameronx,{,||

The lineage relationships and fate of human dendritic cells (DCs) have significance for a number of diseases including HIV where

both blood and tissue DCs may be infected. We used gene expression profiling of human monocyte and DC subpopulations sorted

directly from blood and skin to define the lineage relationships. We also compared these with monocyte-derived DCs (MDDCs)

and MUTZ3 Langerhans cells (LCs) to investigate their relevance as model skin DCs. Hierarchical clustering analysis showed that

myeloid DCs clustered according to anatomical origin rather than putative lineage. Plasmacytoid DCs formed the most discrete

cluster, but ex vivo myeloid cells formed separate clusters of cells both in blood and in skin. Separate and specific DC populations

could be determined within skin, and the proportion of CD14+ dermal DCs (DDCs) was reduced and CD1a+ DDCs increased

during culture, suggesting conversion to CD1a+-expressing cells in situ. This is consistent with origin of the CD1a+ DDCs from

a local precursor rather than directly from circulating blood DCs or monocyte precursors. Consistent with their use as model skin

DCs, the in vitro–derived MDDC and MUTZ3 LC populations grouped within the skin DC cluster. MDDCs clustered most closely

to CD14+ DDCs; furthermore, common unique patterns of C-type lectin receptor expression were identified between these two cell

types. MUTZ3 LCs, however, did not cluster closely with ex vivo–derived LCs. We identified differential expression of novel genes

in monocyte and DC subsets including genes related to DC surface receptors (including C-type lectin receptors, TLRs, and

galectins). The Journal of Immunology, 2013, 190: 000–000.

Dendritic cells (DCs) are a family of professional APCsthat form an important link between the innate and adap-

tive immune systems. They are found as specific subsets

in tissue and blood, and are of either myeloid or plasmacytoid

origin. In their immature form, blood and tissue myeloid DCs

bind foreign Ags by an array of C-type lectin receptors (CLRs)

expressed on their surface. After exposure to foreign Ags or pro-

inflammatory cytokines, DCs mature and migrate to the draining

lymph nodes to present MHC class II–bound foreign Ag to, and

activate, T cells. Plasmacytoid DCs (pDCs) are found mainly in the

blood and lymph nodes, and function primarily to provide antiviral

defense by secretion of very large quantities of IFN-a after mi-

gration to areas of foreign Ag exposure or inflammation, although

in this setting, they can also present Ag and activate T cells (1).

Myeloid DCs can be further divided into functional subsets basedon anatomical distribution and the expression of cell surface mark-

ers. Classical blood myeloid DCs express CD11c and CD1c

(BDCA1), and a CD141 (BDCA3)-expressing subset equivalent to

mouse CD8+ DCs has also been defined (2). DC-like blood cells

that express CD16 and M-DC8 (3, 4) have been recently classified

within the monocyte population (2), although it is clear that there

are distinct functional differences (5). In skin, there are at least

three DC subsets: two found within the dermis that express either

CD1a or CD14, and an epidermal Langerhans cell (LC) expressing

CD1a. In mice, there is an additional langerin-expressing dermal

DC (DDC) that expresses CD103 (2), but no human counterpart

has yet been identified. It is likely that, in time, these DC subsets

will be further divided based on the discovery of new novel ex-

pression markers.

*Westmead Millennium Institute, Westmead, New South Wales 2145, Australia;†Florey Neuroscience Institutes, The University of Melbourne, Melbourne, Victoria3010, Australia; ‡Department of Infectious Diseases, Monash University, Melbourne,Victoria 3004, Australia; xInfectious Diseases Unit, Alfred Hospital Melbourne, Mel-bourne, Victoria 3010, Australia; {Centre for Virology, Burnet Institute, Melbourne,Victoria 3010, Australia; and ||Department of Immunology, Monash University, Mel-bourne, Victoria 3004, Australia

1A.N.H. and C.R.B. contributed equally to this work.

Received for publication March 16, 2012. Accepted for publication October 21,2012.

This work was supported by National Health and Medical Research Council ProgramGrant 358399.

A.N.H. conducted all microarray and quantitative PCR experiments and generatedmonocyte-derived dendritic cells with technical assistance from S.K.M.; A.N.H. car-ried out all experiments using purified ex vivo dermal dendritic cells, generated genelists from the Illumina expression data, and jointly prepared the manuscript; C.R.B.conducted analysis of all microarray data including the construction of dendrograms,heat maps, and the principal component analysis and also jointly prepared the man-uscript; N.N. generated the MUTZ3 Langerhans cells and monocyte-derived Lang-erhans cells and helped in generating ex vivo dermal dendritic cells; K.J.S. conducted

flow cytometric analysis of TLR expression on blood cells; M.K. carried out immu-nofluorescent staining of dermal dendritic cells in foreskin explants and helped withmanuscript revisions; R.A.B. assisted with the generation of monocyte-derived Lang-erhans cells and their processing for Illumina bead array hybridization; S.R.L. pro-vided intellectual input and helped with manuscript preparation; A.L.C. providedintellectual input and helped with manuscript preparation; and P.U.C. isolated allex vivo subsets and jointly prepared the manuscript.

Address correspondence and reprint requests to Dr. Paul U. Cameron at the currentaddress: Department of Infectious Diseases, Monash University, Commercial Road,Melbourne, VIC 3004, Australia. E-mail address: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this article: CLEC, C-type lectin receptor domain familymember; CLR, C-type lectin receptor; DC, dendritic cell; DDC, dermal DC; LC,Langerhans cell; LGALS, lectin, galactoside-binding soluble; MDDC, monocyte-derived DC; MR, mannose receptor; pDC, plasmacytoid DC; PRR, pattern recognitionreceptor; QPCR, quantitative PCR; RF10, RPMI 1640 supplemented with 10% humanAB serum; SIGN, specific intercellular adhesion molecule-3-grabbing nonintegrin.

Copyright� 2012 by TheAmerican Association of Immunologists, Inc. 0022-1767/12/$16.00

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Although the hemopoietic origin of DCs is clear, the preciserelationship of circulating precursors to tissue DCs and the ontogenyof tissue and blood DCs is less well defined in humans. Skin DCsubsets have been proposed to originate from both monocyte pre-cursors and committed local DC precursors largely based on thedifference in murine skin DCs in wild type mice and those deficientfor CSF-1 (6) or its receptor (7), and on human transendothelialmigration models (8, 9). Common myeloid precursors have beenidentified in mice (10, 11), and some studies of human and mouseskin have suggested that CD14+ monocytes are the direct pre-cursors of epidermal LCs (12). It has also been suggested thatCD16+ blood DCs, including those expressing the marker M-DC8(4), may be immediate precursors of some tissue DCs (8, 13).However, reconstitution of skin DCs after bone marrow trans-plantation (14) has suggested that LCs are replenished from long-lived, locally proliferating precursors, and that CD1a+ DDCs arisefrom precursors distinct from LCs (15, 16). The relationship be-tween the human CD11c+ CD1c+ blood myeloid DCs as precursorsof tissue DC subpopulations has been unclear, but in murinemodels, the development of committed DC precursors in bloodand tissue from common myeloid precursors and differentiationin lymphoid tissue is now clear (17).Isolation of ex vivo DC subsets is problematic because they exist

in very low numbers (,1% of human skin and blood), and skinDCs are inherently difficult to isolate as immature cells becausethey are prone to maturation as a result of extraction (18). Forthese reasons, model skin DCs are extensively used for studies ofDC function and viral infection. The most common model,monocyte-derived DCs (MDDCs), can be produced in largenumbers of immature cells by culturing CD14+ monocytes in IL-4and GM-CSF (19, 20). More recently, a model LC has been pro-posed that is derived from the leukemia-derived cell line MUTZ3(21). Although it is proposed that MDDCs and MUTZ3 LCs mostclosely resemble CD14+ DDCs and LCs, respectively, the rele-vance of these two model systems remains unclear.In this study, we aimed to investigate the lineage relationships

between ex vivo–derived blood and skin monocytic and DC subsetscompared with in vitro–derived model MDDCs and MUTZ3 LCs.We primarily used cells directly isolated without culture, but alsoused cells from skin and blood that were cultured for 24 h afterisolation to allow a more direct comparison with the in vitro–cul-tured MDDC and MUTZ3-LC. We initially conducted polygeneticanalysis on gene expression profiles derived by gene arrays of thesecell types. To identify novel markers differentiating between DCsubsets, we then used quantitative PCR (QPCR) to measure geneexpression profiles of surface proteins. We particularly focused onpattern recognition receptors (PRRs) such as TLRs and CLRs be-cause these are involved in specific detection of pathogen mole-cules and are expressed in unique combinations by different cellsubsets to allow them to best recognize pathogens in specificlocations in the body. We also focused on the galectins because theyare known to be expressed by leukocyte subpopulations and play animportant role in the regulation of the immune response (22, 23).

Materials and MethodsIsolation of skin DCs

As previously described (24), skin was separated from s.c. adipose tissueand cut into strips 1.5–2 cm in width. The skin strips were kept in mediumwith antibiotics at 4˚C for 0.5–1 h, and split skin was obtained using a skingraft knife. The split skin was placed in RPMI 1640 (Invitrogen) supple-mented with 10% human AB serum (RF10; Sigma-Aldrich) with 4 mg/mldispase (Worthington) at 4˚C overnight. The split skin was washed in PBSand split into dermis and epidermis using fine forceps. Dermal tissue wascut into 1- to 2-mm blocks using scalpels in a scissoring action, and thedermal blocks were placed in 10 ml RF10 containing 4 mg/ml collagenase

II (Worthington) and incubated at 37˚C with agitation for 1 h. DNase I75 mg/ml (Roche) was added during the last 30 min of incubation and thetissue repeatedly aspirated through a cutoff Pasteur pipette every 5 min.After incubation, the cells were diluted to 50 ml with PBS at room tem-perature and passed through a 70-mm mesh and pelleted. The cell pelletwas washed once in FACS wash (PBS with 1% FCS and 2 mM EDTA) andlabeled with directly conjugated Abs to HLA-DR, CD14, and CD1a. Thehigh HLA-DR–expressing CD14+ and CD1a+ cells were isolated by FACSas previously described (25). The epidermal sheets were either incubatedwith 0.3 mg/ml trypsin in RPMI at 4˚C for 4–6 h and a single-cell sus-pension isolated over a Nycodenz gradient, or treated using a similarmethod to the dermal tissue using collagenase dissociation and DNaselabeled with CD1a and HLA-DR before MACS selection using an auto-MACS Separator and cell sorting for HLA-DR+ CD1a+ cells by flowcytometry. Sorted cells were lysed in guanidinium-containing lysis buffer(Qiagen). When analyzing cells after in vitro culture, we performed theearlier procedure after dermal skin sheets had been cultured in media andemigrating cells collected and included in the analysis.

Isolation of blood mononuclear cell populations and DCs

Isolation of blood mononuclear cell populations and DCs was done as pre-viously described (26). Buffy coats were obtained from the Red Cross BloodTransfusion Service (Sydney and Melbourne, Australia). PBMCs wereisolated over Ficoll Hypaque (GE Healthcare) gradients, and cell pop-ulations were isolated by magnetic bead selection and flow cytometry (Fig.1). In the standard protocol, cells were labeled with mAb to M-DC8(“slan”) (4) and goat anti-mouse IgM beads (Miltenyi), and positively se-lected using MACS columns (Miltenyi). The positively selected cells werelabeled with conjugated Abs to HLA-DR and CD16, and sorted for CD16+,HLA-DR+ large cells by high-speed flow cytometry (FACS Vantage DIVA,FACSAria [BD Bioscience], or MoFlo [Dako Cytomation]). The MACS2

cells were labeled with hybridoma supernatant specific for CD14 [3C10]and CD14+ cells selected using MACS columns. The MACS2 [M-DC82

CD142] fraction was further depleted of lineage markers by labeling withhybridoma supernatant specific for CD3 [OKT3], CD8 [OKT8], CD11b[3G8], and CD19 [FMC63], incubated with MACS GAM-IgG beads. TheMACS+ CD14+ cells were further purified by flow cytometry. The MACS2

[lineage-negative] cells were labeled with fluorescent Abs to HLA-DR,CD123, and CD1c or CD11c before final selection of myeloid DCs(CD1232, CD1c+ CD11c+) and pDCs (CD123+ CD11c2 CD1c2) usingflow cytometry. Cell purity of skin and blood DC populations after cellsorting was between 95 and 99.5% (average 97%), with ,2% contamina-tion with other DC populations. Cultured blood DCs and myeloid cells wereobtained by 24-h culture of the DCs in RF10, IL-3 (10 ng/ml; R&D Sys-tems), and GM-CSF (40 ng/ml; R&D System) to maintain cell viability.

Preparation of in vitro–derived MDDCs and MUTZ3 LCs

MDDCs were differentiated from CD14+ monocytes as previously described(18, 27, 28). Human CD34+ acute myeloid leukemia MUTZ3 cells (providedby S. Santegoets, VU University Medical Centre, Amsterdam, The Nether-lands) were cultured in MEM-a containing ribonucleosides and deoxyribo-nucleosides (Invitrogen) supplemented with 10% conditioned media from thehuman renal carcinoma cell line 5637 and 20% FCS (JRH Biosciences) at105 cells/ml. After 7 d, cells were cultured in MEM-a as described earlier butadditionally supplemented with 100 ng/ml GM-CSF, 2.5 ng/ml TNF-a, and 5ng/ml TGF-b1 (R&D Systems) at 2.53 105 cells/ml. Cells were cultured for10 d to allow differentiation into MUTZ3-derived LCs (MUTZ3 LC). Theculture media were replaced with fresh cytokine-supplemented media on days3 and 7. The phenotype of the differentiated MUTZ3 LCs was assessed byflow cytometry. Immature MUTZ3 LCs were defined as cells that stainedpositively for langerin, CD4, and CD1a, whereas staining negatively for DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (SIGN),mannose receptor (MR), and CD83.

Immunofluorescence microscopy of DDCs

Normal foreskin tissues were obtained from children undergoing circumci-sion, andmechanically separated into inner and outer parts. The inner foreskintissues were snap frozen in OCT, cut into 5-mMsections, placed on slides, andkept at280˚C until used for immunofluorescent staining. Tissues were fixedfor 10 min with ice-cold methanol/acetone (1:1). Foreskin tissues wereblocked with 10% normal goat serum (Sigma-Aldrich) for 30 min at roomtemperature and then incubated for 45 min at 37˚C with rabbit anti-humanDC-SIGN polyclonal Ab (1:50; Abcam) and mouse anti-human CD14 mAb(1:20; BioLegend). After the incubation with the primary Abs, Alexa Fluor546–conjugated goat anti-rabbit (1:400; Molecular Probes) and Alexa Fluor647–conjugated goat anti-mouse (1:200; Molecular Probes) Abs were addedas secondary Abs followed by incubation for 45 min at 37˚C. Tissue was

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incubated for 45 min at 37˚C with FITC-conjugated mouse anti-humanCD1a Ab (1:10; BioLegend). All washes between steps were carried outin PBS. All Abs were diluted in Protein Block Serum-Free solution (DAKO).After staining, ProLong Gold Antifade reagent with DAPI (Invitrogen) wasadded to the stained tissues and coverslips were mounted onto tissue-loadedslides. Slides were visualized through a 340 1.35 NA oil-immersion lenswith an inverted Olympus IX-70 microscope (DeltaVision Image RestorationMicroscope; Applied Precision/Olympus) and a Photometrics CoolSnap QEcamera.

Microarray hybridization and data analysis

Total RNA was extracted from purified cell populations from individualdonors and processed for hybridization to 1 of 55 cDNA gene arrays (HumanResGen 8k; Australian Genome Research Facility) using a common MDDCreference, or 24 bead arrays (sentrix human 6 v2 expression chips; Illumina,San Diego, CA). The RNA extraction, labeling, hybridization, data pro-cessing, and analysis procedures are described previously for the cDNA genearray (18) and Illumina arrays (27). Clustered data were further processed inPARTEK Genomics Suite (Partek) to exclude genes not showing detectableexpression in .80% of arrays and to remove batch effects. Microarray dataare available through the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32648 and (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32400). Genes differentially ex-pressed in at least one group were identified using ANOVA corrected formultiple testing (step-up false discovery rate, p . 0.05), and the data wereclustered using City-block (arrays) and average Euclidean (genes) algo-rithms or by principle components analysis.

Generation and analysis of gene lists

Differential expression analysis was initially conducted in Bead Studiousing the mean of each cell group as the reference. Gene lists were thengenerated by filtering the data set for genes absent in the reference group(detection p . 0.05) and genes present in the DC subset group of interest(detection p , 0.01). The SD of the average signal between biologicalreplicates was then calculated. The SD was then divided by the meanaverage signal to generate a coefficient of variation. Any genes that did notmeet the coefficient of variation cutoff were then removed (1.4 for cell

sets with 2 biological replicates, 1.7 for 3 biological replicates, and 2.0 for4 replicates).

QPCR

QPCR was performed on cDNA derived from the same samples used formicroarray analysis, as well as for additional samples using GAPDH as aninternal reference for normalization using the methods previously described(27, 28). The primer sequences are listed in Supplemental Table I.

TLR expression on cell subsets by flow cytometry

Murine mAbs TLR1-PE (GD2.F4), TLR2-FITC (TL2.1), TLR3-PE (TLR3.7),and TLR4-PE (HTA125) were purchased from eBioscience. UnconjugatedTLR5 mAb (19D759.2), biotinylated TLR6 mAb (86B1153.2), unconjugatedrabbit polyclonal TLR7, TLR8-FITC (44C143), and TLR9-FITC (26C593.2)were purchased from Imgenex. Goat anti-mouse IgG-PE was from Molec-ular Probes, streptavidin-PE from BD Pharmingen, and goat anti-rabbit Ig-FITC from Sigma-Aldrich. Cells were stained for surface expression ofTLR1, TLR2, TLR4, TLR5, and TLR6 or else fixed and permeabilized withCytofix/Cytoperm (BD Biosciences), then stained for intracellular expressionof TLR3, TLR7, TLR8, and TLR9. TLR5, TLR6, and TLR7 Abs weredetected with goat anti-mouse IgG-PE, streptavidin-PE, and goat anti-rabbitIg-FITC, respectively.

ResultsIsolation of ex vivo blood and skin cells and in vitro–derivedDCs

To investigate the lineage relationships between human DC andmonocytic cell populations, we isolated DCs and monocytes fromhuman blood and skin using magnetic bead and flow cytometry–based cell sorting (Fig. 1). We obtained representatives of the cellsrecognized in current classifications (2) and divided the blood cellpopulations into four groups: 1) CD14+ monocytes, 2) CD16/M-DC8+ monocytes/DCs, 3) CD11c+ blood myeloid or classical

FIGURE 1. Isolation of ex vivo blood and skin

cell populations. (Left) blood mononuclear cells

were obtained from normal blood donors as buffy

coat by Ficoll-Hypaque gradients. The M-DC8/

CD16+ DC/monocytes and CD14+ monocytes

were isolated by positive selection using MACS

separation and sorting by flow cytometry. The DC-

enriched populations were isolated by negative

selection using a mixture of Abs to lineage mark-

ers, and the DCs sorted for CD1c+/CD11c+ mye-

loid DCs and CD11c2/BDCA2+ pDCs. (Right)

Skin DCs were isolated from normal split skin by

separation of dermis and epidermis by dispase

treatment at 4˚C and subsequent collagenase/DNase

treatment of dermal and epidermal sheets. The

isolated single cells were then pre-enriched by

MACS separation and sorted by flow cytometry for

CD1a+ epidermal LCs and for separate CD1a+ and

CD14+ DDCs.

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DCs, and 4) CD123+ blood pDCs. Skin DCs were divided intothree groups: 1) CD1a-expressing DDCs, 2) CD14-expressingDDCs (24, 29), and 3) CD1a-expressing epidermal LCs. We alsogenerated the model MDDCs and MUTZ3 LCs in vitro to comparewith the ex vivo–derived skin DCs.

Clustering analysis of isolated cell populations

We first compared different ex vivo DC and monocytic populationsusing hierarchical cluster analysis on gene expression profilesdetected using cDNA microarrays. Hierarchical clustering usesunique subsets of genes expressed in each sample to determine thedegree of similarity between the cell types, providing a represen-tation of the relationship between the samples (Fig. 2A). As ex-pected, pDCs clustered away from the myeloid populations asa distinct unrelated group. The remaining myeloid populationsseparated into two broad clusters. The first contained all blood cellpopulations: CD11c+ myeloid DCs and CD16+ and CD14+ mono-cytes. The second broad cluster contained mixed populations ofskin DCs, comprising the CD14+ and CD1a+ DDCs, as well asepidermal LCs.

Principle component analysis of the expression profiles (Fig. 2B)confirmed the strong separation of blood and skin cells into twobroad groups with contained subgroups. In addition, within theskin DC populations, the CD14+ DDCs and LCs clustered sepa-rately, with the CD1a+ DDC cluster overlapping the two pop-ulations (not seen in the hierarchical clustering). In addition, in theprinciple components analysis, cultured cell populations fell intotwo distinct groups. Cultured CD14+ DDCs clustered closely tothe skin DC populations, whereas the cultured pDCs and bloodmyeloid DCs clustered with the blood cell populations (individualcultured cell types are shown in Supplemental Fig. 1).To further discriminate between the three skin DC subsets and

the blood myeloid DC and monocytic cell populations, the clus-tering analysis was repeated using data generated from Illuminabead arrays that contain complete coverage of the genome. To allowfor a comparison of ex vivo– and in vitro–derived DCs, additionalpopulations were profiled, including MDDCs (a model for CD14+

DDCs) and MUTZ3 LCs (a model for epidermal LCs). In agree-ment with the cDNA arrays, the three ex vivo myeloid blood cellpopulations clustered together, forming a distinct cluster separate

FIGURE 2. Cluster analysis of DC subsets. Cluster analysis of gene expression profiles from blood and skin DC and monocyte populations. Sorted cells

from skin and blood were analyzed by cDNA microarrays (A, B) or Illumina HT12 gene arrays (C). Cell types with the most similar gene expression profiles

cluster together. (A) and (C) represent hierarchical cluster analysis; (B) represents principle component analysis. CD1a DDC, CD1a+ DDC; CD14 DDC,

CD14+ DDC; CD14 mono, CD14+ monocytes; CD16 mono, CD16+ monocytes; iMDDC, immature MDDC; mDC, blood myeloid DC; mMDDC, mature

MDDC; reference, immature MDDC.


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from the skin DCs. The three ex vivo–derived skin DC populationsstill clustered closely, with the CD1a-expressing epidermal LCsand DDCs clustering together and separate to the CD14+ DDCpopulation. Consistent with their use as model skin DCs, thein vitro–derived MDDCs and MUTZ3 LCs both clustered with theskin DC cluster. Immature MDDCs clustered most closely to theCD14+ DDC population. However, the MUTZ3 LCs formed themost discrete cluster, most similar to mature MDDCs, and did notcluster close to the LC populations.

Interconversion of DDC subsets in situ

To explore the lineage relationship of the skin DCs suggested bythe clustering analysis, we looked at DDCs before and after cultureof intact dermis to determine whether there was any evidence ofphenotypic shifts between CD14+ and CD1a+ DDCs in situ. Beforein vitro culture, the high HLA-DR–expressing cells extracted bycollagenase treatment included a similar number of CD14+ DDCscompared with CD1a+ DDCs, but postculture, the CD1a+ DDCswere the predominant population in the emigrant and collagenaseisolated cells (Supplemental Fig. 2A). To further define the dis-tribution of CD14+ and CD1a+ DDCs, we cultured dermal sheetsbefore and after collagenase digestion and compared populationsas shown in Fig. 3A. The comparison populations were: 1) cellsisolated by collagenase digestion at day 0, 2) cells migrating fromday 0 collagenase-treated dermis during overnight culture, 3) cellsisolated from dermis by collagenase after overnight culture, and 4)cells migrating from intact dermal sheets during overnight culture.Total isolated at day 0 cells = (population 1) + (population 2) andat day 1 = (population 3) + (population 4) (Fig. 3D). Phenotypicanalysis of these populations is shown in Fig. 3A. The recovery ofCD1a+ DDCs was greatest during migration from intact dermalsheets, and this population also had the highest frequency ofCD1a+ cells (Fig. 3D). The CD1a+ cells uniformly expressedHLA-DR in directly isolated cells and in migrating cells, makingit unlikely that the increase in CD1a-expressing cells derive froman HLA-DR2 population (Supplemental Fig. 2B). High HLA-DR–expressing CD14+ cells were highest in cells from dermiscollagenase treated at day 0 and in the migrating cells from intactdermis. HLA-DR high, CD14-expressing cells were infrequentamong emigrants from collagenase-treated dermis or isolated bycollagenase treatment at day 1 of culture. Taken together, thesedata suggest that there may be phenotypic conversion or matura-tion of CD14+ DDCs during migration. In intact skin, cells coex-pressing CD14 and CD1a were observed (Fig. 3B, 3C). We nextsorted CD1a+, CD14+, and dual-expressing cells and cultured themovernight as purified populations (Fig. 3B). Conversion of CD14+

to CD1a+ DDCs was not observed; however, cells coexpressingCD14 and CD1a did show increased expression of CD1a andreduced CD14. Taken together, the close clustering by gene ex-pression and the apparent development of increased numbers ofCD1a+ DDCs within cultured explants suggests close lineagerelationships between DDC subpopulations and CD14-expressingcells as precursors for the CD1a-expressing cells within the dermalmicroenvironment in situ.

Comparison of DC surface marker expression

DC and monocytic cell populations express an array of surfacemarkers that are often unique to specific cell subsets. We identifieddifferential expression of these markers from the microarrays, andconfirmed and extended these observations by QPCR using a largersample size. We focused on PRRs, which are involved in specificdetection of pathogen molecules and subsequent signaling. In ad-dition to TLRs and the cytoplasmic RNA binding receptors (RIGI,MDA5, and PKR), we particularly focused on the CLR domain

family members (CLECs) that recognize pathogens and vary in theirexpression on different DC subsets. We also looked at other CLRs(selectins and collectins) and also the galectin family of surfaceproteins (lectin, galactoside-binding soluble [LGALS]), becausethese are known to be expressed by various cells of the immunesystem and participated in immune regulation and homeostasis ofvarious leukocyte subpopulations (22, 23). Finally, we investigatedthe expression of CD1a and genes encoding costimulatory mole-cules indicative of DC maturation (CD40, CD80, CD83, and CD86).The average expression for each cell population is summarized

in Table I and illustrated as a heat map (Fig. 4A). Changes in theexpression of TLR genes in blood-derived cells were confirmed atthe protein level by flow cytometry (TLR1-8; Fig. 4B).CD1a was exclusively expressed in MDDCs, CD1a+ DDCs, and

LCs, consistent with the fact that these are the only cell subsetsthat express this surface marker. LGALS8 was expressed by all celltypes, but other galectins were much more cell-type specific.LGALS2 expression was specific to skin cells. High LGALS1 ex-pression was observed only in DDCs. DDCs were the only cellsthat did not express LGALS9. LGALS7 was expressed only in LCs,which also expressed much lower levels of LGALS4, especiallywhen compared with DDCs. Expression of LGALS12 (GRIP1)was restricted to CD14+ monocytes and LGALS10 (CLC) ex-pression was only in pDCs and MDDCs.Similarly, many of the CLRs were discriminatory for particular

cell subsets. First, known patterns of expression were verified suchas exclusive expression of CLEC4K (langerin) by LCs (Fig. 4C)and CLEC4C (BDCA2) by pDCs (30).Recently, langerin-expressing DDCs have been identified in

mice (31), but it is unclear whether human dermis contains thesecells. We therefore examined the QPCR data more closely todetermine whether any other cell populations expressed low levelsof langerin (Fig. 4C). The CD1a+ DDCs expressed the nexthighest langerin levels (though very low and below the cutoff forinclusion in Table I). However, no langerin expression was detectedin CD14+ DDCs. Even lower langerin expression was detected inMDDCs and in five of the eight CD11c+ blood myeloid DC donors.Novel patterns of CLR expression were also defined. CLEC8A

(OLR1) was observed only in skin DCs. As expected, CLEC13D(mannose receptor I) and the highly related CLEC13DL wereexpressed by MDDCs and DDC subsets, as was CLEC3B (TNA),whereas CLEC13E (mannose receptor II) was expressed byMDDCs, CD11c+ blood myeloid DCs, LCs, and at very low levelsby two of five of the CD1a+ DDC samples. CLEC4E (MINCLE)expression was observed only in DDCs, and these cells alsoexpressed higher levels of CLEC4M (LSIGN) than other cell pop-ulations. CLEC13B (DEC205) was not expressed by CD14+ DDCsand at only very low levels by CD1a+ DDCs, which were also theonly cells found not to express CLEC12A (DCAL2). CLEC4G(LSECtin), CLEC4L (DCSIGN), and CLEC14A (EGFR-5) were alluniquely expressed by MDDCs and CD14+ DDCs. CLEC2B wasexclusively not expressed by MDDCs. CLEC5A (MDL1) expres-sion was specific for CD1a+ DDCs and LCs. CLEC6A and CLEC7A(Dectin 2 and 1) were not expressed by pDCs, and CLEC1Awas notexpressed by pDCs and LCs. SELL (CD62L) was not expressed byany skin DC populations or MDDCs, and SELP (CD62) showedvariable expressed in most cells but was not expressed by anyCD1a+ DDC samples. Most collectins were not expressed by anycell populations; however, COLEC12 was expressed very highly byMDDCs and DDCs, and COLEC7 was not expressed by pDCs.Concerning other PRRs, PKR, RIGI, and MDA5 were con-

sistently expressed across all populations, as were TLR1 andTLR6. TLR5 expression was very low in MDDCs and myeloidblood cells, and absent in all other cells. As expected, TLR7 and


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TLR9 were expressed much more highly in pDCs, which werethe only cells that did not express TLR2 and TLR3. TLR4 andTLR8 were not expressed in DDCs, LCs, and pDCs (one of theCD1a+ and CD14+ DDC donors expressed TLR4 and TLR8, re-spectively). Gene and protein expression of TLRs in blood-derived cell subsets was generally consistent, although TLR7was detected at the gene but not protein level in CD14+ mono-cytes. It is of interest that TLR3 is expressed much more highly onthe model MDDCs and TLR4 on CD14+ monocytes than the othercell populations.

Consistent with the literature, the ex vivo–derived skin DCs allshowed evidence of partial maturation as evidenced by increasedexpression of CD40, CD83, and CD86 (32). These results aresummarized at the top of Table II.

Identification of other genes uniquely expressed in DCsubpopulations

We next examined the Illumina gene expression data to identifygenes whose expression was unique to specific DC populations.Those with the most restricted expression were tested by QPCR

FIGURE 3. Flow cytometric analysis of

cultured dermal DCs. (A) Experimental flow-

chart and phenotyping of DDCs. DDCs were

either directly isolated by collagenase treat-

ment or collected after migrating after over-

night culture and phenotyped for HLA-DR,

CD14, and CD1a expression. The comparison

populations were: 1) cells isolated by collage-

nase digestion at day 0, 2) cells migrating from

intact dermal sheets after overnight culture, 3)

cells migrating from collagenase-treated der-

mis that was then cultured overnight, and 4)

cells isolated by collagenase digestion of intact

dermis that had been cultured overnight. (B)

Cells directly isolated after collagenase diges-

tion of intact dermal sheets at day 0 were

sorted and collected for 1) CD14+ CD1a2, 2)

CD1a+ CD142, and 3) CD1a+ CD14+. Then

they were cultured overnight as single-cell

populations. The phenotype of the cultured

cells was then determined by flow cytometry.

(C) Coexpression of CD14 (FITC) and CD1a

(Alexa Fluor 647) on DDCs in situ. Dermis

was examined for cells expressing CD14 and

CD1a. Scale bar, 20 mM. (D) Recovery of

CD1a+ cells isolated after migration or colla-

genase treatment. The total number of CD1a+

cells isolated normalized against the recovery

of cells from dermis that was collagenase

treated at day 0 for three donors is shown. D1

indicates recovery of cells from dermis col-

lagenase treated after 1 d. Total cells is the

total of cells isolated by migration and by

collagenase treatment. The proportion (%) of

CD1a-expressing cells in each population is

shown in the lower panel.


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Table

I.Monocyte

andDCsubsetsgeneexpressionprofiles

GeneNam

eGeneSymbol

Other

Nam

e(s)

MDDC

CD14+Mono

CD16+Mono

CD11c+

BloodDC

pDC

CD1a+

DDC

CD14+DDC

LC

CD1amolecule

CD1a

CD1,FCB6

+++

——

——

+++

—+++

lectin,galactoside-binding,soluble,1

LGALS1

GAL1,HBL

++

++

++++

+++

+lectin,galactoside-binding,soluble,2

LGALS2

HL14

—+

—+

—+++

++

++


LGALS3

GAL3,CBP35

+++

++

++

++

—+++

+++


LGALS4

GAL4,L36LBP

++

+++

+++++

+++

++++++

330

321


LGALS7

GAL7

——

——

——

—1046


LGALS8

GAL8,PCTA1

+++

+++

+++

+++

++++

++

++

+++


LGALS9

HUAT

++

+++

+++

++

+++

——

+++


LGALS10

GAL10,

CLC

++++

——

—437

——

—lectin,galactoside-binding,soluble,12

LGALS12

GAL12,

GRIP1

—++

——

——

——

C-typelectin

domainfamily1,mem

ber

ACLEC1A

+++

++

+++

+++

—+++

+++

—C-typelectin

domainfamily1,mem

ber

BCLEC1B

——

——

——

——

C-typelectin

domainfamily2,mem

ber

ACLEC2A

KACL

——

——

——

——

C-typelectin

domainfamily2,mem

ber

BCLEC2B

AICL

—+++

+++

+++

++++

++

+++

++

C-typelectin

domainfamily2,mem

ber

CCLEC2Ca

CD69

+++

+++

+++

++++

++++++

++++

++++

+++++

C-typelectin

domainfamily2,mem

ber

LCLEC2La

——

——

——

——

C-typelectin

domainfamily3,mem

ber

ACLEC3Aa

CLECSF1

——

——

——

——

C-typelectin

domainfamily3,mem

ber

BCLEC3B

TNA

++++

——

——

+++++

+++

—C-typelectin

domainfamily4,mem

ber

ACLEC4A

DCIR

+++

++

++

++++

+++

+C-typelectin

domainfamily4,mem

ber

CCLEC4C

BDCA2,CD303

——

——

120

——

—C-typelectin

domainfamily4,mem

ber

DCLEC4D

MCL

—++++++

+++++/2

+++++/2

—+++++/2

+++++

—C-typelectin

domainfamily4,mem

ber

ECLEC4E

MIN

CLE

—+

——

—+++

+++

—C-typelectin

domainfamily4,mem

ber

FCLEC4F

KCLR

++++

+893

++++/2

—++++++

++

340

C-typelectin

domainfamily4,mem

ber

GCLEC4G

LSECtin

++

——

——

++++

—C-typelectin

domainfamily4,mem

ber

JCLEC4Ja

FCER2

+++++

+++++

++++++

—++

+++

+C-typelectin

domainfamily4,mem

ber

KCLEC4K

Langerin,CD207

——

——

——

—+++++

C-typelectin

domainfamily4,mem

ber

LCLEC4L

DCSIG

N,CD209

+++

——

——

—++

—C-typelectin

domainfamily4,mem

ber

MCLEC4M

LSIG

N,CD299

++

+++

++

++++++

+++

++

C-typelectin

domainfamily4,mem

ber

H1

CLEC4H1a

ASGR1

++

+++

+++

+++

+++

++

+C-typelectin

domainfamily4,mem

ber

H2

CLEC4H2a

ASGR2

+++

+++++

++

+++

++++

+++

+++

C-typelectin

domainfamily5,mem

ber

ACLEC5A

MDL1

——

——

—++++

++++

C-typelectin

domainfamily5,mem

ber

BCLEC5Ba

KLRB1

++

+++

+++

++

++

+++

C-typelectin

domainfamily5,mem

ber

CCLEC5Ca

KLRF1

++

++

++

++

+C-typelectin

domainfamily6,mem

ber

ACLEC6A

CLEC4N,DECTIN

2++

++

+++

—379

+++

++++

C-typelectin

domainfamily7,mem

ber

ACLEC7A

BGR,DECTIN

1+++

+++

+++

+—

+++

++++

+++

C-typelectin

domainfamily8,mem

ber

ACLEC8A

OLR1,LOX1

138

——

——

379

423

173

C-typelectin

domainfamily9,mem

ber

ACLEC9A

DNGR1

++/2

+/2

++++/2

—+++++/2

—+++++/2

C-typelectin

domainfamily10,mem

ber

ACLEC10A

HML,CD301

++

——

++

—+++

++

+C-typelectin

domainfamily11,mem

ber

ACLEC11Aa

LSLC1,SCGF

++

+++

++

++

++

++

++

C-typelectin

domainfamily12,mem

ber

ACLEC12A

DCAL-2,MICL,CLL1

++++

+++

++

+++

—++++

+++/2

C-typelectin

domainfamily12,mem

ber

BCLEC12B

—+++++

++++

+++

++++

++++

++++

—C-typelectin

domainfamily13,mem

ber

ACLEC13A

DCL1,CD302

+++

++++

+++++

++++

+++++

+++++

+++

+++

C-typelectin

domainfamily13,mem

ber

BCLEC13B

DEC205,LY75,CD205

+++

++

+++

+++

+++

+—

++++

C-typelectin

domainfamily13,mem

ber

CCLEC13Ca

PLA2R1

——

——

——

——

C-typelectin

domainfamily13,mem

ber

DCLEC13D

MCR1,CD206

+++++

——

——

998

5464

—C-typelectin

domainfamily13,mem

ber

Dlike

CLEC13DLa

MRC1L1

+++

——

——

++

++

—C-typelectin

domainfamily13,mem

ber

ECLEC13E

MCR2,CD280

++++

——

+++

—+/2

—+++

C-typelectin

domainfamily14,mem

ber

ACLEC14A

CEG1,EGFR-5

+++

——

——

—+++++

+/2

C-typelectin

domainfamily15,mem

ber

ACLEC15Aa

KLRG1

++

++

++

+++

++

++

+C-typelectin

domainfamily15,mem

ber

BCLEC15Ba

KLRG2

—+

++

——

——

(Table

continues)


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Table

I.(Continued

) GeneNam

eGeneSymbol

Other

Nam

e(s)

MDDC

CD14+Mono

CD16+Mono

CD11c+

BloodDC

pDC

CD1a+

DDC

CD14+DDC

LC

C-typelectin

domainfamily16,mem

ber

ACLEC16A

+++

++

+++

++

+++

++

+++++

C-typelectin-like1

CLECL1

DCAL1

++

—+

++

+++++

+++

++

selectin

ESELEa

CD62E,ELAM,

——

—+

—+

——

selectin

LSELL

CD62L,LAM1,

LEU8

—++++

+++

+++

+++++

——

—selectin

PSELP

CD62,LECAM3

+++/2

++/2

+/2

++++/2

—++++

+++

collectin1

COLEC1a

MBL2

——

——

——

——

collectin4

COLEC4a

SFTPA

1—

——

——

——

—collectin5

COLEC5a

SFTPA

2—

——

——

——

—collectin7

COLEC7

SFTPD

++++

415

++++

—407

++++

125

collectin10

COLEC10a

CLL1

——

——

——

——

collectin11

COLEC11a

CLK1

——

——

——

——

collectin12

COLEC12

CLP1

++++++

+++

—++

—124

377

+++

TLR1

TLR1

CD281

++++

+++

+++

+++

++++

+++

+++

++++

TLR2

TLR2

CD282

+++

+++

+++

++

—+++

+++

++/2

TLR3

TLR3

CD283

+++

++

++++

—+++

+++

+++

TLR4

TLR4

CD284,ARMD10

++++

+++

+++

++

—++/2

——

TLR5

TLR5

SLEB1

++

++

——

——

TLR6

TLR6

CD286

++

++

++

+++

+++

+++

+++

+++

TLR7

TLR7

++++

+++

++

++++++

+++

++

+++

TLR8

TLR8

CD288

++++

+++++

+++++

+++

——

+/2

TLR9

TLR9

CD289

++

—+++

114

——

—TLR10

TLR10

CD290

——

++

++++

—++

protein

kinaseRNA

activated

PKR

EIF2A

K2

+++

++

+++

++

+++

+++

+++

+++

retinoic

acid–induciblegeneI

RIG

IDDX58

+++

+++

++++

++

++

++

++

++

melanomadifferentiation–associated

protein

5MDA5

IFIH

1+

++

++

++

++

++

CD40

molecule

CD40

TNFRSF5,CDW40

+—

—+

—++

++

++

CD80

molecule

CD80

BB1,CD28LG

+—

——

—+

—+

CD83

molecule

CD83

BL11,HB15

+++

++

++++

+++

+++

CD86

molecule

CD86

B70,CD28LG2

++

+++

+++

++

++

+++

+++

Cellulargeneexpressionwas

determined

inbloodandskin

cellpopulationsbyQPCR

norm

alized

toGAPDH

expressionexceptwhereindicated.

Theam

ountofrelative

expressionofeach

geneissummarized

asfollow

s:++++++=,25;+++++=10–24.9;++++=5.0–9.9;+++=1–4.9;++=0.50–0.99;+=0.10–0.49;(2

)=.0.1;+/2

=expressed

only

insomepopulations.Im

mature

MDDCs(n

=4),CD14+monocytes(m

ono;n=10),CD16+mono(n

=11),bloodmDCs(n

=8),pDCs(n

=10),CD1a+

DDCs(n

=4),CD14+DDCs(n

=4),andLCs(n

=4).Expressiondataforindividualsamplesareillustratedas

aheatmap

(see

Fig.4A).

aExpressiondataapproxim

ated

from

Illuminabeadarraydataandnotconfirm

edbyQPCR

(thesegenes

werenondiscrim

inatory

andincluded

sothefullrangeofCLRsiscovered).


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(Fig. 5). We identified specificity of expression for the followinggenes and cell types: CCL18 in MDDCs, SLC11A1 in all mono-cytes, SMARCD3 in CD14+ monocytes, LYPD2 and PTPN3 inCD16+ monocytes, SLA2 in CD1a+ DDCs, PRSS2 and PEEP1 inCD14+ DDCs, and EFNB3 in LCs. In addition, we identified lackof expression for the following genes and cell types: TNI2 not inMDDCs, INF2 not in CD16+ monocytes, ACOT7 not in myeloidblood cells, STK11 not in blood myeloid DCs, PLXNC1 not inCD1a+ DDCs, and PKN not in LCs. These results are summarizedat the bottom of Table II.

DiscussionFunctional clustering of genetic markers represents a way ofstudying lineage and differentiation of highly purified cell pop-ulations (33). Microarray analysis remains one of the most pow-erful tools for the identification of such lineage relationshipsespecially among blood cells and malignancies (34–38). In thisstudy, we have used hierarchical clustering and principle compo-

nent analysis to further understand the origins and relationshipsbetween isolated blood and skin DC and monocytic cell pop-ulations (Fig. 1). We have gone on to identify novel markers to helpfurther distinguish between different cell populations using QPCR.Hierarchical clustering and principle component analysis (Fig.

2A, 2B) showed that pDCs formed the expected discrete clusterbecause of well-established early lineage differences from othermyeloid cells. The myeloid skin and blood populations alsoclustered separately, indicating no clear similarity of gene ex-pression profiles between these two general cell populations.These data did not provide direct support for the model of CD11c+

blood myeloid DCs (or a subset of them) as direct precursors toskin DCs in the resting state. However, treatment of blood CD14+

monocytes in vitro with cytokines differentiates them into MDDCsthat cluster with skin DCs demonstrating the plasticity of theselineages. Principle component analysis and Illumina expressionarray data (Fig. 2B, 2C) revealed individual subsets within thesetwo main populations that clustered apart from each other. Within

FIGURE 4. Analysis of PRR expression. (A) Expression of genes encoding PRRs on different cell populations. Genes encoding PRRs that were de-

termined to be differentially expressed by microarray were confirmed and extended by QPCR. The expression of each gene was compared with GAPDH

expression by QPCR on the same sample. The log10 of the fold change relative to GAPDH was then used to generate the heat map. (B) Expression of TLR

proteins on different cell populations. TLR surface and intracellular expression was determined by flow cytometry. The histogram shows the average ratio

of the mean fluorescence intensities (MFIs) of the sample: isotype control with SE bars (n = 9). (C) Langerin expression levels in cell populations. mRNA

expression levels were determined by QPCR. Data are represented as a box-and-whisker plot. The line in the center of each box denotes the median value; the

lower and upper edges of the box, the 25th and 75th percentiles, respectively; and the whiskers, the maximum and minimum values. Immature MDDCs (n = 4),

CD14+ monocytes (mono; n = 10), CD16+ mono (n = 11), blood mDCs (n = 8), pDCs (n = 10), CD1a+ DDCs (n = 4), CD14+ DDCs (n = 4), and LCs (n = 4).


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the skin, the CD1a+ DDCs and LCs clustered most closely, con-sistent with the common expression of CD1a on their surface, withthe CD14+ DDCs forming a separate cluster. When we investi-gated the effects of skin explant culture on isolated DDCs in situ,we saw a phenotypic shift from CD14-expressing cells to CD1a-expressing cells, with some cells expressing both markers (Fig.3C, 3D). In addition, we found that after overnight culture, theproportion of CD1a+ cells isolated increased and CD14+ cellsdecreased. These observations of close clustering of skin DCs anda phenotypic shift after culture suggest that the dermal skin pop-ulations may be related and interconvert from CD14+ to CD1a+

DDCs without direct origin from separate blood monocytic pre-

cursors. However, we did not observe a conversion of CD14+

to CD1a+ cells after purification, indicating that a factor withinthe microenvironment of the skin is necessary to drive the conver-sion. Our findings in this study of expression profiles of segregatedpopulations based on anatomical location (blood versus skin) areconsistent with a recent murine DC microarray study that lookedat lymphoid resident DCs (39).Recently, langerin-expressing CD103+ DDCs have been dis-

covered in mice (31). Langerin-expressing DDCs have also beenreported in human skin; however, these have been considered asepidermal LCs in transit through the dermis (40), and there re-mains speculation as to the existence of an equivalent population

Table II. Summary of cell subset discriminatory genes

Gene Name Gene Symbol Other Name(s) Cell-Type SpecificityRemark Relative toOther Cell Types

lectin, galactoside-binding, soluble, 1 LGALS1 GAL1, HBL DDC Highly expressedlectin, galactoside-binding, soluble, 2 LGALS2 HL14 Skin DC Moderately expressedlectin, galactoside-binding, soluble, 4 LGALS4 GAL4, L36LBP Epidermal LC Low expressionlectin, galactoside-binding, soluble, 7 LGALS7 GAL7 Epidermal LC Very highly expressedlectin, galactoside-binding, soluble, 9 LGALS9 HUAT DDC Exclusive nonexpressionlectin, galactoside-binding, soluble, 10 LGALS10 CLC Blood pDC/MDDC High expressionlectin, galactoside-binding, soluble, 12 LGALS12 GAL12, GRIP1 CD14+ mono Exclusive expressionC-type lectin domain family 1, member A CLEC1A Blood pDC/epidermal LC Exclusive nonexpressionC-type lectin domain family 2, member B CLEC2B AICL MDDC Exclusive nonexpressionC-type lectin domain family 3, member B CLEC3B TN DDC/MDDC Exclusive expressionC-type lectin domain family 4, member C CLEC4C BDCA2, CD303 Blood pDC Exclusive high expressionC-type lectin domain family 4, member E CLEC4E MINCLE DDC Exclusive high expressionC-type lectin domain family 4, member G CLEC4G LSECtin CD14+ DDC/MDDC Exclusive expressionC-type lectin domain family 4, member K CLEC4K langerin, CD207 Epidermal LC Exclusive high expressionC-type lectin domain family 4, member L CLEC4L DCSIGN, CD209 CD14+ DDC/MDDC Exclusive expressionC-type lectin domain family 4, member M CLEC4M LSIGN, CD299 CD1a+ DDC Highly expressedC-type lectin domain family 5, member A CLEC5A MDL1 CD1a+ DDC/epidermal LC Exclusive expressionC-type lectin domain family 6, member A CLEC6A DECTIN-2, CLEC4N Blood pDC Exclusive nonexpressionC-type lectin domain family 7, member A CLEC7A DECTIN-1, BGR Blood pDC Exclusive nonexpressionC-type lectin domain family 8, member A CLEC8A OLR1, LOX1 Skin DC Exclusive expressionC-type lectin domain family 12, member A CLEC12A MICL, DCAL-2, CLL1 CD1A+ DDC Exclusive nonexpressionC-type lectin domain family 13, member B CLEC13B DEC205, LY75, CD205 DDC NonexpressionC-type lectin domain family 13, member D CLEC13D MRC1, CD206 DDC/MDDC Exclusive expressionC-type lectin domain family 13, member D-like CLEC13DLa MRC1L1 DDC/MDDC Exclusive expressionC-type lectin domain family 14, member A CLEC14A EGFR-5, CEG1 CD14+ DDC/MDDC Exclusive expressionselectin L SELL CD62L, LAM1 Blood cells Exclusive expressionselectin P SELP CD62 CD1A+ DDC Exclusive nonexpressioncollectin 7 COLEC7 SFTPD Blood pDC Exclusive nonexpressioncollectin 12 COLEC12 CLP1 DDC/MDDC Very high expressionTLR 2 TLR2 CD282 Blood pDC Exclusive nonexpressionTLR 3 TLR3 CD283 Blood pDC Exclusive nonexpressionTLR 7 TLR7 Blood pDCs Very high expressionTLR 9 TLR9 CD289 Blood pDCs Very high expressionsolute carrier family 11 (proton-coupled

divalent metal ion transporters), member 1SLC11A1 LSH, NRAMP CD14+/CD16+ mono Exclusive expression

SWI/SNF-related, matrix-associated,actin-dependent regulator of chromatin,subfamily d, member 3

SMARCD3 BAF60C CD14+ mono Highly expressed

solute carrier family 5(sodium/glucose cotransporter),member 10

SLC5A10 SGLT5 CD16+ mono Exclusive expression

LY6/PLAUR domain containing 2 LYPD2 LYPDC2 CD16+ mono Highly expressedprotein tyrosine phosphatase,

nonreceptor type 3PTPN3 PTPH1 CD16+ mono Highly expressed

inverted formin, FH2 and WH2 domaincontaining

INF2 CD16+ mono Exclusive nonexpression

serine/threonine kinase 11 STK11 LKB1, PJS mDC Exclusive nonexpressionSrc-like adaptor 2 SLA2 SLAP2, MARS CD1a+ DDC Exclusive expressionreceptor accessory protein 1 REEP1 CD14+ DDC Exclusive expressionplexin C1 PLXNC1 CD232, VESPR CD14+ DDC/MDDC Exclusive nonexpressionephrin-B3 EFNB3 EFL6, EPLG8, LERK8 Epidermal LC Exclusive expressionprotein kinase N1 PKN PAK1, PRK1 Epidermal LC Exclusive nonexpressionchemokine (C-C motif) ligand 18 CCL18 AMAC1 MDDC Exclusive expressiontroponin I type 2 (skeletal, fast) TNNI2 AMCD2B, DA2B, FSSV MDDC Exclusive nonexpression

Summary of genes whose expression or nonexpression has been identified to discriminate between cell subsets.mDC, CD11c+ blood myeloid DC; mono, monocyte.


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in humans. Although we found that LCs were the only cell pop-ulation that expressed high langerin levels, we did find that CD1a+

DDCs (but not CD14+ DDCs) also expressed langerin, albeitat very low levels (Fig. 4C). We cannot completely exclude the

possibility of minor contamination between CD1a-expressing LCsand DDCs. In addition, the principle components analysis showedan overlap between the CD1a+ DDC and LC populations, whichalso clustered closely together in the Illumina expression analysis.

FIGURE 5. Determination of cell subset discriminating gene markers. QPCR analysis was carried out to confirm the gene expression of key markers

selected to be specific in their (A) expression or (B) nonexpression to particular cell subsets identified by Illumina microarray. The histograms show the

relative expression compared with GAPDH with SE bars. MDDCs (n = 4), CD14+ monocytes (mono; n = 10), CD16+ mono (n = 11), mDCs (n = 8), pDCs

(n = 9), CD1a+ DDCs (n = 4), CD14+ DDCs (n = 4), and LCs (n = 4).


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This suggests that a human langerin-expressing DDC subset mayexist within the CD1a+ DDC population but represent only a tracepopulation in resting skin.The differentiation of CD14+ monocytes into MDDCs using IL-

4 and GM-CSF (19, 20) has provided a method for generatinglarge numbers of DCs for immunotherapy and a model for thestudy of DC biology in vitro. Similarly, LC-like cells can begenerated in vitro either from CD34+ bone marrow or bloodprecursors cultured in GM-CSF and TNF-a (41), or from CD14+

monocytes using TGF-b, GM-CSF, and IL-4 (42). Although hu-man monocytes have been shown to differentiate into both mac-rophages and DCs after transendothelial migration (13), and aTGF-b–dependent pathway from CD14+ monocytes to LCs mayexist in vivo (12), it is unclear what the in vivo equivalents of thesemodel DCs are. MDDCs have been proposed to most closely re-semble CD14+ DDCs partly because both express the CLR DC-SIGN (25).LC-like cells derived from CD34+ or CD14+ precursors are

assumed to mimic LCs because of their high langerin expression,but unlike LCs, they also express the CLRs DC-SIGN and MR,similar to MDDCs. In addition to expressing high levels of lan-gerin, MUTZ3 LCs lack the expression of DC-SIGN and MR (21),and for this reason, we chose to investigate this model LC. Insupport of their use as model skin DCs, both the MDDC andMUTZ3 LC cell populations did reside within the general skin DCcluster (Fig. 2C). Consistent with their proposed similarity toCD14+ DDCs, the immature MDDCs clustered most closely tothis ex vivo cell type. However, the MUTZ3 LCs cells did notcluster closely to LCs and rather formed the most discrete clusterwithin the skin cells alongside mature MDDCs, indicating thatcaution should be applied to findings derived from this modelsystem. However, they would remain a useful model for studiesinvolving surface receptors.In support ofMDDCs asmodel DDCs, we found common patterns

of exclusive CLR expression, both uniquely expressing CLEC13D(mannose receptor), CLEC13DL, CLEC3B (TNA), and COLEC12.Furthermore, in support of a closer similarity to CD14+ DDCs thanCD1a+ DDCs, we confirmed the finding that CLEC4L (DCSIGN)expression was restricted to CD14+ DDCs and MDDCs, and wealso show that the related CLEC4G (LSECtin) and CLEC14A werealso uniquely expressed by these two cell types. Like DC-SIGN,LSECtin is involved in pathogen binding and recognizes endoge-nous activated T cells (43), and CLEC14A has recently been iden-tified as an endothelial marker involved in cell migration (44). NoCLRs were uniquely expressed or nonexpressed by MDDCs andLCs or CD1a+ DDCs.In this study, we have identified additional specific markers for the

different DC/monocyte subpopulations (Table II), which may serveas a basis to more easily distinguish between subpopulations: 1)CD14+ and CD16+ blood monocytes specifically express SLC11A1,SLC5A10, and SMARCD3. SLC11A1 and SLC5A10 are both solutecarrier proteins. SLC5A10 has been shown to be exclusively ex-pressed in myeloid lineage cells and plays a role in macrophagedifferentiation where the recruitment of SWI/SNF complex is re-quired for its transcriptional activation (45). SMARCD3 is a corecomponent of the nuclear SWI/SNF complex linking the cell-type–specific function of the proteins encoded by these two genes. Thiscomplex is also required for HIV TAT-driven transcriptional acti-vation (46). 2) CD14+ monocytes uniquely express LGALS12,previously been shown to be involved in regulating the cell cycle(47). 3) CD16+ monocytes uniquely express LYPD2 and PTPN. Nostudies have been published on LYPD2, but PTPN3 is a member ofthe protein tyrosine phosphatase family that regulates a variety ofcellular processes including cell growth and differentiation. INF2

was exclusively not expressed by CD16+ monocyte. INF2 is in-volved in immunologic synapse formation (48), and its non-expression is consistent with limited ability of CD16+ monocytes toform immunological synapses compared with DCs or other cellsthat form such synapses. 4) CD11c+ blood myeloid DCs did notexclusively express any genes. However, they alone did not expressthe serine/threonine kinase STK11. 5) pDCs showed the mostmarked differences in their surface marker expression comparedwith other ex vivo cells as expected. They exclusively expressedvery high levels of CLEC4C (BDCA2), LGALS10 (CLC), TLR7, andTLR9. In addition, they were the only cells not to express COLEC7,TLR2, TLR3, as well as CLEC6A (dectin 2) CLEC7A (dectin 1),which play a role in innate recognition of fungi (49).Skin cell populations uniquely express the low-density lipo-

protein scavenger receptor CLEC8A (OLR1). Selectin L was notexpressed by skin DCs, consistent with an essential role in bloodleukocyte emigration through vascular endothelium (50). Presenceor absence of some markers were specific for skin DC subpop-ulations. 1) Epidermal LCs uniquely express very high levels ofLGALS7 and langerin, as well as EFNB3, as previously shown byothers (51). This is consistent with known expression of LGALS7by epithelial cells (52). LGALS2 was expressed at much lowerlevels than other cells. We found that our uncultured LCs did notexpress TLR4 as previously reported by others (53), and expres-sion of TLR2 was detected in only two of the four LC donors. Thisis consistent with previous work (54), although a recent publica-tion shows TLR2 and TLR4 expression on LCs after culture (55).LCs did not express TLR5 or TLR9 and uniquely lacked PKN,which regulates NF-kB signaling (integral to TLR-mediated sig-nal transduction) via phosphorylation of TRAF1 (56). 2) DDCsuniquely express LGALS1, which plays a role in leukocyte mi-gration, modulates proinflammatory cytokine release (57), andmediates DC anergy (58), and CLEC4E (MINCLE), which playsa role in recognizing pathogenic fungus (59). LGALS9, whichplays a pivotal role in T cell immunity (60), was uniquely notexpressed by DDCs. 3) CD1a+ DDCs uniquely express SLA2,which has recently been shown to regulate DC maturation (61). Inaddition, they expressed much higher levels of CLEC4M (LSIGN)shown to be associated with HIV transmission (62). CD1a+ DDCswere the only cell population that consistently did not expressthe monocytic cell marker CELC12A (DCAL2). 4) CD14+ DDCsexclusively expressed REEP1, which is involved in targetingreceptors to lipid rafts (63). Conversely, PLXNC1 was exclusivelynonexpressed. In murine DCs, PLXNC1 signaling inhibits integ-rins resulting in decreased cell adhesion and migration, and DCsin mice lacking PLXNC1 showed a reduced capacity to stimulateT cells (64).The ontogeny of DCs, and their migration to and population of

tissue have important implications for dissemination of infectiousorganisms. For example, HIV can readily infect or be carried byblood DCs or CD16+ monocytes (26), which marginalize (5) andemigrate under inflammatory conditions to generate tissue DCs.We have previously shown that different CLRs may bind HIV intissue (25), and recent work has suggested that within the skin,langerin-mediated binding by HIV may have differing effectsdepending on the state of the cell (65). In this study, we havenow extended our understanding of DC–HIV interactions byproviding profiles for expression of surface markers on DC sub-sets in blood and skin that may be important in HIV entry andvirus carriage by these DC subsets. Such data are also relevant toother epitheliotropic viruses such as HSV. Understanding theseprocesses can only be achieved by a study of primary cells and useof uniquely expressed and nonexpressed markers as we have iden-tified in this study.


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In conclusion, we show in this study that skin and blood DC/monocyte populations form two distinct polygenetic cell pop-ulations. This and the finding that CD14+ DDCs convert to CD1a+

DDCs in situ does not support the hypothesis that CD11c+ bloodmyeloid DCs represent a direct skin DC precursor, but ratherindicates that CD14+ DDCs may be precursors to CD1a+ DDCs. Inline with their common expression of CD1a, LCs and CD1a+

DDCs had a greater degree of similarity in expression profilescompared with the CD1a-nonexpressing CD14+ DDCs. Our find-ings support the proposal that MDDCs most closely resembleCD14+ DDCs. In addition to DCSIGN, we have identified threeCLRs that these two cells populations uniquely coexpress. MDDCsprobably represent the closest model of DDC, but MUTZ3 LCs donot cluster with LCs and, therefore, may be limited as a LC model.Furthermore, we have identified uniquely expressed and nonex-pressed novel genes that discriminate between DC subsets. Thesefindings now provide a broader range of unique markers for fullydifferentiated human skin and blood DCs, which may help indefining human skin precursors during injury inflammation. Theyalso indicate multiple new directions for research to define furtherbiological differences between DC subsets.

AcknowledgmentsFlow cytometry was performed in the Alfred Monash Research Education

Precinct core flow cytometry unit (Melbourne, Victoria, Australia) and the

Westmead Research hub Flow Cytometry Centre, supported by Westmead

Millennium Institute, Children’s Medical Research Institute, Kid’s Re-

search Institute, National Health and Medical Research Council and Can-

cer Institute New South Wales. Dr. John Anstee (Mercy Hospital, East

Melbourne, Victoria, Australia) and Dr. Norman Oldbourne (Dalcross Ad-

ventist Hospital, Killara, New South Wales, Australia) provided discarded

skin samples from apronectomy operations.

DisclosuresThe authors have no financial conflicts of interest.

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Identification of Lineage Relationships and Novel Markers of Blood and Skin Human Dendritic Cells

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