Dendritic Cells Novel Markers of Blood and Skin Human

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

This information is current as Andrew N. Harman, Chris R. Bye, Najla Nasr, Kerrie J. of September 26, 2021. Sandgren, Min Kim, Sarah K. Mercier, Rachel A. Botting, Sharon R. Lewin, Anthony L. Cunningham and Paul U. Cameron J Immunol 2013; 190:66-79; Prepublished online 26

November 2012; Downloaded from doi: 10.4049/jimmunol.1200779 http://www.jimmunol.org/content/190/1/66

Supplementary http://www.jimmunol.org/content/suppl/2012/11/29/jimmunol.120077 http://www.jimmunol.org/ Material 9.DC1 References This article cites 63 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/190/1/66.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Identiﬁcation of Lineage Relationships and Novel Markers of Blood 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 Downloaded from 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 http://www.jimmunol.org/ 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: 66–79.

endritic cells (DCs) are a family of professional APCs Myeloid DCs can be further divided into functional subsets based that form an important link between the innate and adap- on anatomical distribution and the expression of cell surface mark- D tive immune systems. They are found as specific subsets ers. Classical blood myeloid DCs express CD11c and CD1c in tissue and blood, and are of either myeloid or plasmacytoid (BDCA1), and a CD141 (BDCA3)-expressing subset equivalent to + origin. In their immature form, blood and tissue myeloid DCs mouse CD8 DCs has also been defined (2). DC-like blood cells by guest on September 26, 2021 that express CD16 and M-DC8 (3, 4) have been recently classified bind foreign Ags by an array of C-type lectin receptors (CLRs) within the monocyte population (2), although it is clear that there expressed on their surface. After exposure to foreign Ags or pro- are distinct functional differences (5). In skin, there are at least inflammatory cytokines, DCs mature and migrate to the draining three DC subsets: two found within the dermis that express either lymph nodes to present MHC class II–bound foreign Ag to, and CD1a or CD14, and an epidermal Langerhans cell (LC) expressing activate, T cells. Plasmacytoid DCs (pDCs) are found mainly in the CD1a. In mice, there is an additional langerin-expressing dermal blood and lymph nodes, and function primarily to provide antiviral DC (DDC) that expresses CD103 (2), but no human counterpart defense by secretion of very large quantities of IFN-a after mi- has yet been identified. It is likely that, in time, these DC subsets gration to areas of foreign Ag exposure or inflammation, although will be further divided based on the discovery of new novel ex- in this setting, they can also present Ag and activate T cells (1). pression markers.

*Westmead Millennium Institute, Westmead, New South Wales 2145, Australia; flow cytometric analysis of TLR expression on blood cells; M.K. carried out immu- †Florey Neuroscience Institutes, The University of Melbourne, Melbourne, Victoria nofluorescent staining of dermal dendritic cells in foreskin explants and helped with 3010, Australia; ‡Department of Infectious Diseases, Monash University, Melbourne, manuscript revisions; R.A.B. assisted with the generation of monocyte-derived Lang- Victoria 3004, Australia; xInfectious Diseases Unit, Alfred Hospital Melbourne, Mel- erhans cells and their processing for Illumina bead array hybridization; S.R.L. pro- { bourne, Victoria 3010, Australia; Centre for Virology, Burnet Institute, Melbourne, vided intellectual input and helped with manuscript preparation; A.L.C. provided Victoria 3010, Australia; and ||Department of Immunology, Monash University, Mel- intellectual input and helped with manuscript preparation; and P.U.C. isolated all bourne, Victoria 3004, Australia ex vivo subsets and jointly prepared the manuscript. 1A.N.H. and C.R.B. contributed equally to this work. Address correspondence and reprint requests to Dr. Paul U. Cameron at the current address: Department of Infectious Diseases, Monash University, Commercial Road, Received for publication March 16, 2012. Accepted for publication October 21, Melbourne, VIC 3004, Australia. E-mail address: [email protected] 2012. The online version of this article contains supplemental material. This work was supported by National Health and Medical Research Council Program Grant 358399. Abbreviations used in this article: CLEC, C-type lectin receptor domain family member; CLR, C-type lectin receptor; DC, dendritic cell; DDC, dermal DC; LC, A.N.H. conducted all microarray and quantitative PCR experiments and generated Langerhans cell; LGALS, lectin, galactoside-binding soluble; MDDC, monocyte- monocyte-derived dendritic cells with technical assistance from S.K.M.; A.N.H. car- derived DC; MR, mannose receptor; pDC, plasmacytoid DC; PRR, pattern recognition ried out all experiments using purified ex vivo dermal dendritic cells, generated gene receptor; QPCR, quantitative PCR; RF10, RPMI 1640 supplemented with 10% human lists from the Illumina expression data, and jointly prepared the manuscript; C.R.B. AB serum; SIGN, specific intercellular adhesion molecule-3-grabbing nonintegrin. conducted analysis of all microarray data including the construction of dendrograms, heat maps, and the principal component analysis and also jointly prepared the man- Ó uscript; N.N. generated the MUTZ3 Langerhans cells and monocyte-derived Lang- Copyright 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 erhans cells and helped in generating ex vivo dermal dendritic cells; K.J.S. conducted www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200779 The Journal of Immunology 67

Although the hemopoietic origin of DCs is clear, the precise II (Worthington) and incubated at 37˚C with agitation for 1 h. DNase I relationship of circulating precursors to tissue DCs and the ontogeny 75 mg/ml (Roche) was added during the last 30 min of incubation and the of tissue and blood DCs is less well defined in humans. Skin DC tissue repeatedly aspirated through a cutoff Pasteur pipette every 5 min. After incubation, the cells were diluted to 50 ml with PBS at room tem- subsets have been proposed to originate from both monocyte pre- perature and passed through a 70-mm mesh and pelleted. The cell pellet cursors and committed local DC precursors largely based on the was washed once in FACS wash (PBS with 1% FCS and 2 mM EDTA) and difference in murine skin DCs in wild type mice and those deficient labeled with directly conjugated Abs to HLA-DR, CD14, and CD1a. The + + for CSF-1 (6) or its receptor (7), and on human transendothelial high HLA-DR–expressing CD14 and CD1a cells were isolated by FACS as previously described (25). The epidermal sheets were either incubated migration models (8, 9). Common myeloid precursors have been with 0.3 mg/ml trypsin in RPMI at 4˚C for 4–6 h and a single-cell sus- identified in mice (10, 11), and some studies of human and mouse pension isolated over a Nycodenz gradient, or treated using a similar skin have suggested that CD14+ monocytes are the direct pre- method to the dermal tissue using collagenase dissociation and DNase labeled with CD1a and HLA-DR before MACS selection using an auto- cursors of epidermal LCs (12). It has also been suggested that + + CD16+ blood DCs, including those expressing the marker M-DC8 MACS Separator and cell sorting for HLA-DR CD1a cells by flow cytometry. Sorted cells were lysed in guanidinium-containing lysis buffer (4), may be immediate precursors of some tissue DCs (8, 13). (Qiagen). When analyzing cells after in vitro culture, we performed the However, reconstitution of skin DCs after bone marrow trans- earlier procedure after dermal skin sheets had been cultured in media and plantation (14) has suggested that LCs are replenished from long- emigrating cells collected and included in the analysis. + lived, locally proliferating precursors, and that CD1a DDCs arise Isolation of blood mononuclear cell populations and DCs from precursors distinct from LCs (15, 16). The relationship between the human CD11c+ CD1c+ blood myeloid DCs as precursors Isolation of blood mononuclear cell populations and DCs was done as previously described (26). Buffy coats were obtained from the Red Cross Blood of tissue DC subpopulations has been unclear, but in murine Transfusion Service (Sydney and Melbourne, Australia). PBMCs were Downloaded from models, the development of committed DC precursors in blood isolated over Ficoll Hypaque (GE Healthcare) gradients, and cell pop- and tissue from common myeloid precursors and differentiation ulations were isolated by magnetic bead selection and flow cytometry (Fig. in lymphoid tissue is now clear (17). 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- Isolation of ex vivo DC subsets is problematic because they exist lected using MACS columns (Miltenyi). The positively selected cells were in very low numbers (,1% of human skin and blood), and skin labeled with conjugated Abs to HLA-DR and CD16, and sorted for CD16+, + DCs are inherently difficult to isolate as immature cells because HLA-DR large cells by high-speed flow cytometry (FACS Vantage DIVA, http://www.jimmunol.org/ 2 they are prone to maturation as a result of extraction (18). For FACSAria [BD Bioscience], or MoFlo [Dako Cytomation]). The MACS cells were labeled with hybridoma supernatant specific for CD14 [3C10] these reasons, model skin DCs are extensively used for studies of + 2 2 and CD14 cells selected using MACS columns. The MACS [M-DC8 DC function and viral infection. The most common model, CD142] fraction was further depleted of lineage markers by labeling with monocyte-derived DCs (MDDCs), can be produced in large hybridoma supernatant specific for CD3 [OKT3], CD8 [OKT8], CD11b numbers of immature cells by culturing CD14+ monocytes in IL-4 [3G8], and CD19 [FMC63], incubated with MACS GAM-IgG beads. The + + 2 and GM-CSF (19, 20). More recently, a model LC has been pro- MACS CD14 cells were further purified by flow cytometry. The MACS [lineage-negative] cells were labeled with fluorescent Abs to HLA-DR, posed that is derived from the leukemia-derived cell line MUTZ3 CD123, and CD1c or CD11c before final selection of myeloid DCs (21). Although it is proposed that MDDCs and MUTZ3 LCs most (CD1232,CD1c+ CD11c+) and pDCs (CD123+ CD11c2 CD1c2) using closely resemble CD14+ DDCs and LCs, respectively, the rele- flow cytometry. Cell purity of skin and blood DC populations after cell by guest on September 26, 2021 vance of these two model systems remains unclear. sorting was between 95 and 99.5% (average 97%), with ,2% contamina- In this study, we aimed to investigate the lineage relationships tion with other DC populations. Cultured blood DCs and myeloid cells were obtained by 24-h culture of the DCs in RF10, IL-3 (10 ng/ml; R&D Sys- between ex vivo–derived blood and skin monocytic and DC subsets tems), and GM-CSF (40 ng/ml; R&D System) to maintain cell viability. compared with in vitro–derived model MDDCs and MUTZ3 LCs. Preparation of in vitro–derived MDDCs and MUTZ3 LCs We primarily used cells directly isolated without culture, but also used cells from skin and blood that were cultured for 24 h after MDDCs were differentiated from CD14+ monocytes as previously described + isolation to allow a more direct comparison with the in vitro–cul- (18, 27, 28). Human CD34 acute myeloid leukemia MUTZ3 cells (provided tured MDDC and MUTZ3-LC. We initially conducted polygenetic by S. Santegoets, VU University Medical Centre, Amsterdam, The Nether- lands) were cultured in MEM-a containing ribonucleosides and deoxyribo- analysis on gene expression profiles derived by gene arrays of these nucleosides (Invitrogen) supplemented with 10% conditioned media from the cell types. To identify novel markers differentiating between DC human renal carcinoma cell line 5637 and 20% FCS (JRH Biosciences) at subsets, we then used quantitative PCR (QPCR) to measure gene 105 cells/ml. After 7 d, cells were cultured in MEM-a as described earlier but a expression profiles of surface proteins. We particularly focused on additionally supplemented with 100 ng/ml GM-CSF, 2.5 ng/ml TNF- ,and5 ng/ml TGF-b1(R&DSystems)at2.53 105 cells/ml. Cells were cultured for pattern recognition receptors (PRRs) such as TLRs and CLRs be- 10 d to allow differentiation into MUTZ3-derived LCs (MUTZ3 LC). The cause these are involved in specific detection of pathogen mole- culture media were replaced with fresh cytokine-supplemented media on days cules and are expressed in unique combinations by different cell 3 and 7. The phenotype of the differentiated MUTZ3 LCs was assessed by subsets to allow them to best recognize pathogens in specific flow cytometry. Immature MUTZ3 LCs were defined as cells that stained locations in the body. We also focused on the galectins because they positively for langerin, CD4, and CD1a, whereas staining negatively for DC- specific intercellular adhesion molecule-3-grabbing nonintegrin (SIGN), are known to be expressed by leukocyte subpopulations and play an mannose receptor (MR), and CD83. important role in the regulation of the immune response (22, 23). Immunofluorescence microscopy of DDCs Materials and Methods Normal foreskin tissues were obtained from children undergoing circumci- Isolation of skin DCs sion, and mechanically separated into inner and outer parts. The inner foreskin tissues were snap frozen in OCT, cut into 5-mM sections, placed on slides, and As previously described (24), skin was separated from s.c. adipose tissue kept at 280˚C until used for immunofluorescent staining. Tissues were fixed and cut into strips 1.5–2 cm in width. The skin strips were kept in medium for 10 min with ice-cold methanol/acetone (1:1). Foreskin tissues were with antibiotics at 4˚C for 0.5–1 h, and split skin was obtained using a skin blocked with 10% normal goat serum (Sigma-Aldrich) for 30 min at room graft knife. The split skin was placed in RPMI 1640 (Invitrogen) supple- temperature and then incubated for 45 min at 37˚C with rabbit anti-human mented with 10% human AB serum (RF10; Sigma-Aldrich) with 4 mg/ml DC-SIGN polyclonal Ab (1:50; Abcam) and mouse anti-human CD14 mAb dispase (Worthington) at 4˚C overnight. The split skin was washed in PBS (1:20; BioLegend). After the incubation with the primary Abs, Alexa Fluor and split into dermis and epidermis using fine forceps. Dermal tissue was 546–conjugated goat anti-rabbit (1:400; Molecular Probes) and Alexa Fluor cut into 1- to 2-mm blocks using scalpels in a scissoring action, and the 647–conjugated goat anti-mouse (1:200; Molecular Probes) Abs were added dermal blocks were placed in 10 ml RF10 containing 4 mg/ml collagenase as secondary Abs followed by incubation for 45 min at 37˚C. Tissue was 68 LINEAGE RELATIONSHIP IN HUMAN DENDRITIC CELLS incubated for 45 min at 37˚C with FITC-conjugated mouse anti-human sets with 2 biological replicates, 1.7 for 3 biological replicates, and 2.0 for CD1a Ab (1:10; BioLegend). All washes between steps were carried out 4 replicates). in PBS. All Abs were diluted in Protein Block Serum-Free solution (DAKO). After staining, ProLong Gold Antifade reagent with DAPI (Invitrogen) was QPCR added to the stained tissues and coverslips were mounted onto tissue-loaded slides. Slides were visualized through a 340 1.35 NA oil-immersion lens QPCR was performed on cDNA derived from the same samples used for with an inverted Olympus IX-70 microscope (DeltaVision Image Restoration microarray analysis, as well as for additional samples using GAPDH as an Microscope; Applied Precision/Olympus) and a Photometrics CoolSnap QE internal reference for normalization using the methods previously described camera. (27, 28). The primer sequences are listed in Supplemental Table I. Microarray hybridization and data analysis TLR expression on cell subsets by flow cytometry Total RNA was extracted from purified cell populations from individual Murine mAbs TLR1-PE (GD2.F4), TLR2-FITC (TL2.1), TLR3-PE (TLR3.7), donors and processed for hybridization to 1 of 55 cDNA gene arrays (Human and TLR4-PE (HTA125) were purchased from eBioscience. Unconjugated ResGen 8k; Australian Genome Research Facility) using a common MDDC TLR5 mAb (19D759.2), biotinylated TLR6 mAb (86B1153.2), unconjugated reference, or 24 bead arrays (sentrix human 6 v2 expression chips; Illumina, rabbit polyclonal TLR7, TLR8-FITC (44C143), and TLR9-FITC (26C593.2) San Diego, CA). The RNA extraction, labeling, hybridization, data pro- were purchased from Imgenex. Goat anti-mouse IgG-PE was from Molec- cessing, and analysis procedures are described previously for the cDNA gene ular Probes, streptavidin-PE from BD Pharmingen, and goat anti-rabbit Ig- array (18) and Illumina arrays (27). Clustered data were further processed in FITC from Sigma-Aldrich. Cells were stained for surface expression of PARTEK Genomics Suite (Partek) to exclude genes not showing detectable TLR1, TLR2, TLR4, TLR5, and TLR6 or else fixed and permeabilized with expression in .80% of arrays and to remove batch effects. Microarray data Cytofix/Cytoperm (BD Biosciences), then stained for intracellular expression are available through the Gene Expression Omnibus database (http://www. of TLR3, TLR7, TLR8, and TLR9. TLR5, TLR6, and TLR7 Abs were ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32648 and (http://www.ncbi. detected with goat anti-mouse IgG-PE, streptavidin-PE, and goat anti-rabbit Downloaded from nlm.nih.gov/geo/query/acc.cgi?acc=GSE32400). Genes differentially ex- Ig-FITC, respectively. pressed in at least one group were identified using ANOVA corrected for multiple testing (step-up false discovery rate, p . 0.05), and the data were clustered using City-block (arrays) and average Euclidean (genes) algo- Results rithms or by principle components analysis. Isolation of ex vivo blood and skin cells and in vitro–derived Generation and analysis of gene lists DCs

Differential expression analysis was initially conducted in Bead Studio To investigate the lineage relationships between human DC and http://www.jimmunol.org/ using the mean of each cell group as the reference. Gene lists were then monocytic cell populations, we isolated DCs and monocytes from generated by filtering the data set for genes absent in the reference group human blood and skin using magnetic bead and flow cytometry– (detection p . 0.05) and genes present in the DC subset group of interest based cell sorting (Fig. 1). We obtained representatives of the cells (detection p , 0.01). The SD of the average signal between biological recognized in current classifications (2) and divided the blood cell replicates was then calculated. The SD was then divided by the mean + average signal to generate a coefficient of variation. Any genes that did not populations into four groups: 1) CD14 monocytes, 2) CD16/M- meet the coefficient of variation cutoff were then removed (1.4 for cell DC8+ monocytes/DCs, 3) CD11c+ blood myeloid or classical by guest on September 26, 2021

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 markers, and the DCs sorted for CD1c+/CD11c+ myeloid 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. The Journal of Immunology 69

DCs, and 4) CD123+ blood pDCs. Skin DCs were divided into Principle component analysis of the expression profiles (Fig. 2B) three groups: 1) CD1a-expressing DDCs, 2) CD14-expressing confirmed the strong separation of blood and skin cells into two DDCs (24, 29), and 3) CD1a-expressing epidermal LCs. We also broad groups with contained subgroups. In addition, within the generated the model MDDCs and MUTZ3 LCs in vitro to compare skin DC populations, the CD14+ DDCs and LCs clustered sepa- with the ex vivo–derived skin DCs. rately, with the CD1a+ DDC cluster overlapping the two populations (not seen in the hierarchical clustering). In addition, in the Clustering analysis of isolated cell populations principle components analysis, cultured cell populations fell into We first compared different ex vivo DC and monocytic populations two distinct groups. Cultured CD14+ DDCs clustered closely to using hierarchical cluster analysis on gene expression profiles the skin DC populations, whereas the cultured pDCs and blood detected using cDNA microarrays. Hierarchical clustering uses myeloid DCs clustered with the blood cell populations (individual unique subsets of genes expressed in each sample to determine the cultured cell types are shown in Supplemental Fig. 1). degree of similarity between the cell types, providing a represen- To further discriminate between the three skin DC subsets and tation of the relationship between the samples (Fig. 2A). As ex- the blood myeloid DC and monocytic cell populations, the clus- pected, pDCs clustered away from the myeloid populations as tering analysis was repeated using data generated from Illumina a distinct unrelated group. The remaining myeloid populations bead arrays that contain complete coverage of the genome. To allow separated into two broad clusters. The first contained all blood cell for a comparison of ex vivo– and in vitro–derived DCs, additional populations: CD11c+ myeloid DCs and CD16+ and CD14+ mono- populations were profiled, including MDDCs (a model for CD14+ cytes. The second broad cluster contained mixed populations of DDCs) and MUTZ3 LCs (a model for epidermal LCs). In agree- skin DCs, comprising the CD14+ and CD1a+ DDCs, as well as ment with the cDNA arrays, the three ex vivo myeloid blood cell Downloaded from epidermal LCs. populations clustered together, forming a distinct cluster separate http://www.jimmunol.org/ by guest on September 26, 2021

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. 70 LINEAGE RELATIONSHIP IN HUMAN DENDRITIC CELLS from the skin DCs. The three ex vivo–derived skin DC populations family members (CLECs) that recognize pathogens and vary in their still clustered closely, with the CD1a-expressing epidermal LCs expression on different DC subsets. We also looked at other CLRs and DDCs clustering together and separate to the CD14+ DDC (selectins and collectins) and also the galectin family of surface population. Consistent with their use as model skin DCs, the proteins (lectin, galactoside-binding soluble [LGALS]), because in vitro–derived MDDCs and MUTZ3 LCs both clustered with the these are known to be expressed by various cells of the immune skin DC cluster. Immature MDDCs clustered most closely to the system and participated in immune regulation and homeostasis of CD14+ DDC population. However, the MUTZ3 LCs formed the various leukocyte subpopulations (22, 23). Finally, we investigated most discrete cluster, most similar to mature MDDCs, and did not the expression of CD1a and genes encoding costimulatory mole- cluster close to the LC populations. cules indicative of DC maturation (CD40, CD80, CD83,andCD86). The average expression for each cell population is summarized Interconversion of DDC subsets in situ in Table I and illustrated as a heat map (Fig. 4A). Changes in the To explore the lineage relationship of the skin DCs suggested by expression of TLR genes in blood-derived cells were confirmed at the clustering analysis, we looked at DDCs before and after culture the protein level by flow cytometry (TLR1-8; Fig. 4B). of intact dermis to determine whether there was any evidence of CD1a was exclusively expressed in MDDCs, CD1a+ DDCs, and phenotypic shifts between CD14+ and CD1a+ DDCs in situ. Before LCs, consistent with the fact that these are the only cell subsets in vitro culture, the high HLA-DR–expressing cells extracted by that express this surface marker. LGALS8 was expressed by all cell collagenase treatment included a similar number of CD14+ DDCs types, but other galectins were much more cell-type specific. compared with CD1a+ DDCs, but postculture, the CD1a+ DDCs LGALS2 expression was specific to skin cells. High LGALS1 ex- were the predominant population in the emigrant and collagenase pression was observed only in DDCs. DDCs were the only cells Downloaded from isolated cells (Supplemental Fig. 2A). To further define the dis- that did not express LGALS9. LGALS7 was expressed only in LCs, tribution of CD14+ and CD1a+ DDCs, we cultured dermal sheets which also expressed much lower levels of LGALS4, especially before and after collagenase digestion and compared populations when compared with DDCs. Expression of LGALS12 (GRIP1) as shown in Fig. 3A. The comparison populations were: 1) cells was restricted to CD14+ monocytes and LGALS10 (CLC) ex- isolated by collagenase digestion at day 0, 2) cells migrating from pression was only in pDCs and MDDCs.

day 0 collagenase-treated dermis during overnight culture, 3) cells Similarly, many of the CLRs were discriminatory for particular http://www.jimmunol.org/ isolated from dermis by collagenase after overnight culture, and 4) cell subsets. First, known patterns of expression were verified such cells migrating from intact dermal sheets during overnight culture. as exclusive expression of CLEC4K (langerin) by LCs (Fig. 4C) Total isolated at day 0 cells = (population 1) + (population 2) and and CLEC4C (BDCA2) by pDCs (30). at day 1 = (population 3) + (population 4) (Fig. 3D). Phenotypic Recently, langerin-expressing DDCs have been identified in analysis of these populations is shown in Fig. 3A. The recovery of mice (31), but it is unclear whether human dermis contains these CD1a+ DDCs was greatest during migration from intact dermal cells. We therefore examined the QPCR data more closely to sheets, and this population also had the highest frequency of determine whether any other cell populations expressed low levels CD1a+ cells (Fig. 3D). The CD1a+ cells uniformly expressed of langerin (Fig. 4C). The CD1a+ DDCs expressed the next HLA-DR in directly isolated cells and in migrating cells, making highest langerin levels (though very low and below the cutoff for by guest on September 26, 2021 it unlikely that the increase in CD1a-expressing cells derive from inclusion in Table I). However, no langerin expression was detected an HLA-DR2 population (Supplemental Fig. 2B). High HLA- in CD14+ DDCs. Even lower langerin expression was detected in DR–expressing CD14+ cells were highest in cells from dermis MDDCs and in five of the eight CD11c+ blood myeloid DC donors. collagenase treated at day 0 and in the migrating cells from intact Novel patterns of CLR expression were also defined. CLEC8A dermis. HLA-DR high, CD14-expressing cells were infrequent (OLR1) was observed only in skin DCs. As expected, CLEC13D among emigrants from collagenase-treated dermis or isolated by (mannose receptor I) and the highly related CLEC13DL were collagenase treatment at day 1 of culture. Taken together, these expressed by MDDCs and DDC subsets, as was CLEC3B (TNA), data suggest that there may be phenotypic conversion or matura- whereas CLEC13E (mannose receptor II)wasexpressedby tion of CD14+ DDCs during migration. In intact skin, cells coex- MDDCs, CD11c+ blood myeloid DCs, LCs, and at very low levels pressing CD14 and CD1a were observed (Fig. 3B, 3C). We next by two of five of the CD1a+ DDC samples. CLEC4E (MINCLE) sorted CD1a+,CD14+, and dual-expressing cells and cultured them expression was observed only in DDCs, and these cells also overnight as purified populations (Fig. 3B). Conversion of CD14+ expressed higher levels of CLEC4M (LSIGN) than other cell pop- to CD1a+ DDCs was not observed; however, cells coexpressing ulations. CLEC13B (DEC205) was not expressed by CD14+ DDCs CD14 and CD1a did show increased expression of CD1a and and at only very low levels by CD1a+ DDCs, which were also the reduced CD14. Taken together, the close clustering by gene ex- only cells found not to express CLEC12A (DCAL2). CLEC4G pression and the apparent development of increased numbers of (LSECtin), CLEC4L (DCSIGN), and CLEC14A (EGFR-5)wereall CD1a+ DDCs within cultured explants suggests close lineage uniquely expressed by MDDCs and CD14+ DDCs. CLEC2B was relationships between DDC subpopulations and CD14-expressing exclusively not expressed by MDDCs. CLEC5A (MDL1) expres- cells as precursors for the CD1a-expressing cells within the dermal sion was specific for CD1a+ DDCs and LCs. CLEC6A and CLEC7A microenvironment in situ. (Dectin 2 and 1) were not expressed by pDCs, and CLEC1A was not expressed by pDCs and LCs. SELL (CD62L) was not expressed by Comparison of DC surface marker expression any skin DC populations or MDDCs, and SELP (CD62) showed DC and monocytic cell populations express an array of surface variable expressed in most cells but was not expressed by any markers that are often unique to specific cell subsets. We identified CD1a+ DDC samples. Most collectins were not expressed by any differential expression of these markers from the microarrays, and cell populations; however, COLEC12 was expressed very highly by confirmed and extended these observations by QPCR using a larger MDDCs and DDCs, and COLEC7 was not expressed by pDCs. sample size. We focused on PRRs, which are involved in specific Concerning other PRRs, PKR, RIGI,andMDA5 were con- detection of pathogen molecules and subsequent signaling. In ad- sistently expressed across all populations, as were TLR1 and dition to TLRs and the cytoplasmic RNA binding receptors (RIGI, TLR6. TLR5 expression was very low in MDDCs and myeloid MDA5, and PKR), we particularly focused on the CLR domain blood cells, and absent in all other cells. As expected, TLR7 and The Journal of Immunology 71

FIGURE 3. Flow cytometric analysis of cultured dermal DCs. (A)Experimentalﬂow- chart and phenotyping of DDCs. DDCs were either directly isolated by collagenase treatment or collected after migrating after overnight culture and phenotyped for HLA-DR, CD14, and CD1a expression. The comparison populations were: 1) cells isolated by collagenase digestion at day 0, 2) cells migrating from intact dermal sheets after overnight culture, 3) Downloaded from cells migrating from collagenase-treated dermis 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 digestion of intact dermal sheets at day 0 were sorted and collected for 1) CD14+ CD1a2,2) http://www.jimmunol.org/ 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- by guest on September 26, 2021 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 collagenase 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.

TLR9 wereexpressedmuchmorehighlyinpDCs,whichwere Consistent with the literature, the ex vivo–derived skin DCs all the only cells that did not express TLR2 and TLR3. TLR4 and showed evidence of partial maturation as evidenced by increased TLR8 were not expressed in DDCs, LCs, and pDCs (one of the expression of CD40, CD83, and CD86 (32). These results are CD1a+ and CD14+ DDC donors expressed TLR4 and TLR8,re- summarized at the top of Table II. spectively). Gene and protein expression of TLRs in blood- derived cell subsets was generally consistent, although TLR7 Identification of other genes uniquely expressed in DC was detected at the gene but not protein level in CD14+ mono- subpopulations cytes. It is of interest that TLR3 is expressed much more highly on We next examined the Illumina gene expression data to identify the model MDDCs and TLR4 on CD14+ monocytes than the other genes whose expression was unique to specific DC populations. cell populations. Those with the most restricted expression were tested by QPCR Table I. Monocyte and DC subsets gene expression profiles 72

Gene Name Gene Symbol Other Name(s) MDDC CD14+ Mono CD16+ Mono CD11c+ Blood DC pDC CD1a+ DDC CD14+ DDC LC CD1a molecule CD1a CD1, FCB6 +++ — — — — +++ — +++ lectin, galactoside-binding, soluble, 1 LGALS1 GAL1, HBL + + + + + +++ +++ + lectin, galactoside-binding, soluble, 2 LGALS2 HL14 — + — + — +++ ++ ++ lectin, galactoside-binding, soluble, 3 LGALS3 GAL3, CBP35 +++ ++ ++ ++ — +++ +++ + lectin, galactoside-binding, soluble, 4 LGALS4 GAL4, L36LBP ++ +++ +++++ +++ ++++++ 330 321 + lectin, galactoside-binding, soluble, 7 LGALS7 GAL7 —— — — — — — 1046 lectin, galactoside-binding, soluble, 8 LGALS8 GAL8, PCTA1 +++ +++ +++ +++ ++++ ++ ++ +++ lectin, galactoside-binding, soluble, 9 LGALS9 HUAT ++ +++ +++ ++ +++ — — +++ lectin, galactoside-binding, soluble, 10 LGALS10 GAL10, CLC ++++ — — — 437 — — — lectin, galactoside-binding, soluble, 12 LGALS12 GAL12, GRIP1 — ++ — — ———— C-type lectin domain family 1, member A CLEC1A +++ ++ +++ +++ — +++ +++ — C-type lectin domain family 1, member B CLEC1B —— — — ———— C-type lectin domain family 2, member A CLEC2A KACL —— — — ———— C-type lectin domain family 2, member B CLEC2B AICL — +++ +++ +++ ++++ ++ +++ ++ C-type lectin domain family 2, member C CLEC2Ca CD69 +++ +++ +++ ++++ ++++++ ++++ ++++ +++++ C-type lectin domain family 2, member L CLEC2La —— — — —— — — C-type lectin domain family 3, member A CLEC3Aa CLECSF1 —— — — ———— C-type lectin domain family 3, member B CLEC3B TNA ++++ — — — — +++++ +++ — C-type lectin domain family 4, member A CLEC4A DCIR +++ + + ++ + +++ +++ + C-type lectin domain family 4, member C CLEC4C BDCA2, CD303 — — — — 120 — — — C-type lectin domain family 4, member D CLEC4D MCL — ++++++ +++++/2 +++++/2 — +++++/2 +++++ — C-type lectin domain family 4, member E CLEC4E MINCLE — + — — — +++ +++ — C-type lectin domain family 4, member F CLEC4F KCLR ++++ + 893 ++++/2 — ++++++ ++ 340 C-type lectin domain family 4, member G CLEC4G LSECtin ++ — — — — ++++ — C-type lectin domain family 4, member J CLEC4Ja FCER2 +++++ +++++ + +++++ — ++ +++ + C-type lectin domain family 4, member K CLEC4K Langerin, CD207 — — — — — — — +++++ CELLS DENDRITIC HUMAN IN RELATIONSHIP LINEAGE C-type lectin domain family 4, member L CLEC4L DCSIGN, CD209 +++ — — — — — ++ — C-type lectin domain family 4, member M CLEC4M LSIGN, CD299 ++ + ++ ++ + +++++ +++ ++ C-type lectin domain family 4, member H1 CLEC4H1a ASGR1 ++ +++ +++ +++ + ++ ++ + C-type lectin domain family 4, member H2 CLEC4H2a ASGR2 +++ +++++ ++ +++ + +++ +++ +++ C-type lectin domain family 5, member A CLEC5A MDL1 —— — — — ++++ + +++ C-type lectin domain family 5, member B CLEC5Ba KLRB1 ++ + ++ + ++ ++ ++ +++ C-type lectin domain family 5, member C CLEC5Ca KLRF1 ++ + + + + + + + C-type lectin domain family 6, member A CLEC6A CLEC4N, DECTIN2 ++ ++ + ++ — 379 +++ ++++ C-type lectin domain family 7, member A CLEC7A BGR, DECTIN 1 +++ +++ +++ + — +++ ++++ +++ C-type lectin domain family 8, member A CLEC8A OLR1, LOX1 138 — — — — 379 423 173 C-type lectin domain family 9, member A CLEC9A DNGR1 + +/2 +/2 ++++/2 — +++++/2 — +++++/2 C-type lectin domain family 10, member A CLEC10A HML, CD301 ++ — — ++ — +++ ++ + C-type lectin domain family 11, member A CLEC11Aa LSLC1, SCGF ++ + ++ ++ ++ ++ ++ ++ C-type lectin domain family 12, member A CLEC12A DCAL-2, MICL, CLL1 + +++ +++ ++ +++ — ++++ +++/2 C-type lectin domain family 12, member B CLEC12B — +++++ ++++ +++ ++++ ++++ ++++ — C-type lectin domain family 13, member A CLEC13A DCL1, CD302 +++ ++++ +++++ ++++ +++++ +++++ +++ +++ C-type lectin domain family 13, member B CLEC13B DEC205, LY75, CD205 +++ ++ +++ +++ +++ + — ++++ C-type lectin domain family 13, member C CLEC13Ca PLA2R1 —— — — ———— C-type lectin domain family 13, member D CLEC13D MCR1, CD206 +++++ — — — — 998 5464 — C-type lectin domain family 13, member D like CLEC13DLa MRC1L1 +++ — — — — ++ ++ — C-type lectin domain family 13, member E CLEC13E MCR2, CD280 ++++ — — +++ — +/2 —+++ C-type lectin domain family 14, member A CLEC14A CEG1, EGFR-5 +++ — — — — — +++++ +/2 C-type lectin domain family 15, member A CLEC15Aa KLRG1 ++ + + ++ +++ ++ ++ + C-type lectin domain family 15, member B CLEC15Ba KLRG2 — + + + ————

(Table continues)

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Gene Name Gene Symbol Other Name(s) MDDC CD14+ Mono CD16+ Mono CD11c+ Blood DC pDC CD1a+ DDC CD14+ DDC LC C-type lectin domain family 16, member A CLEC16A +++ ++ +++ ++ +++ ++ + ++++ C-type lectin-like 1 CLECL1 DCAL1 ++ — + ++ + ++++ +++ ++ selectin E SELEa CD62E, ELAM, — — — + — + —— selectin L SELL CD62L, LAM1, LEU8 — ++++ +++ +++ +++++ — — — selectin P SELP CD62,LECAM3 + ++/2 ++/2 +/2 ++++/2 — ++++ +++ collectin 1 COLEC1a MBL2 —— — — ———— collectin 4 COLEC4a SFTPA1 —— — — ———— collectin 5 COLEC5a SFTPA2 —— — — ———— collectin 7 COLEC7 SFTPD + +++ 415 ++++ — 407 ++++ 125 collectin 10 COLEC10a CLL1 —— — — ———— collectin 11 COLEC11a CLK1 —— — — ———— collectin 12 COLEC12 CLP1 ++++++ +++ — ++ — 124 377 +++ TLR 1 TLR1 CD281 ++++ +++ +++ +++ ++++ +++ +++ ++++ TLR 2 TLR2 CD282 +++ +++ +++ ++ — +++ +++ ++/2 TLR 3 TLR3 CD283 +++ ++ + +++ — +++ +++ +++ TLR 4 TLR4 CD284, ARMD10 ++++ +++ +++ ++ — ++/2 —— TLR 5 TLR5 SLEB1 + + + + ———— TLR 6 TLR6 CD286 ++ ++ ++ +++ +++ +++ +++ +++ TLR 7 TLR7 + +++ +++ ++ ++++++ +++ ++ +++ TLR 8 TLR8 CD288 ++++ +++++ +++++ +++ — — +/2 TLR 9 TLR9 CD289 + + — +++ 114 — — — TLR 10 TLR10 CD290 —— + + + +++ — ++ protein kinase RNA activated PKR EIF2AK2 +++ ++ +++ ++ +++ +++ +++ +++ retinoic acid–inducible gene I RIGI DDX58 +++ +++ ++++ ++ ++ ++ ++ ++ melanoma differentiation–associated protein 5 MDA5 IFIH1 + ++ ++ + + + + ++ CD40 molecule CD40 TNFRSF5, CDW40 + — — + — ++ ++ ++ CD80 molecule CD80 BB1, CD28LG + — — — — + — + CD83 molecule CD83 BL11, HB15 + ++ + + + +++ +++ +++ CD86 molecule CD86 B70, CD28LG2 ++ +++ +++ ++ ++ + ++ +++ Cellular gene expression was determined in blood and skin cell populations by QPCR normalized to GAPDH expression except where indicated. The amount of relative expression of each gene is summarized as follows: ++++++ = ,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 in some populations. 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). Expression data for individual samples are illustrated as a heat map (see Fig. 4A). aExpression data approximated from Illumina bead array data and not conﬁrmed by QPCR (these genes were nondiscriminatory and included so the full range of CLRs is covered).

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

(Fig. 5). We identified specificity of expression for the following nent analysis to further understand the origins and relationships genes and cell types: CCL18 in MDDCs, SLC11A1 in all mono- between isolated blood and skin DC and monocytic cell pop- cytes, SMARCD3 in CD14+ monocytes, LYPD2 and PTPN3 in ulations (Fig. 1). We have gone on to identify novel markers to help CD16+ monocytes, SLA2 in CD1a+ DDCs, PRSS2 and PEEP1 in further distinguish between different cell populations using QPCR. CD14+ DDCs, and EFNB3 in LCs. In addition, we identified lack Hierarchical clustering and principle component analysis (Fig. of expression for the following genes and cell types: TNI2 not in 2A, 2B) showed that pDCs formed the expected discrete cluster MDDCs, INF2 not in CD16+ monocytes, ACOT7 not in myeloid because of well-established early lineage differences from other blood cells, STK11 not in blood myeloid DCs, PLXNC1 not in myeloid cells. The myeloid skin and blood populations also CD1a+ DDCs, and PKN not in LCs. These results are summarized clustered separately, indicating no clear similarity of gene ex- at the bottom of Table II. pression profiles between these two general cell populations. These data did not provide direct support for the model of CD11c+ Discussion blood myeloid DCs (or a subset of them) as direct precursors to Functional clustering of genetic markers represents a way of skin DCs in the resting state. However, treatment of blood CD14+ studying lineage and differentiation of highly purified cell pop- monocytes in vitro with cytokines differentiates them into MDDCs ulations (33). Microarray analysis remains one of the most pow- that cluster with skin DCs demonstrating the plasticity of these erful tools for the identification of such lineage relationships lineages. Principle component analysis and Illumina expression especially among blood cells and malignancies (34–38). In this array data (Fig. 2B, 2C) revealed individual subsets within these study, we have used hierarchical clustering and principle compo- two main populations that clustered apart from each other. Within The Journal of Immunology 75

Table II. Summary of cell subset discriminatory genes

Remark Relative to Gene Name Gene Symbol Other Name(s) Cell-Type Speciﬁcity Other Cell Types lectin, galactoside-binding, soluble, 1 LGALS1 GAL1, HBL DDC Highly expressed lectin, galactoside-binding, soluble, 2 LGALS2 HL14 Skin DC Moderately expressed lectin, galactoside-binding, soluble, 4 LGALS4 GAL4, L36LBP Epidermal LC Low expression lectin, galactoside-binding, soluble, 7 LGALS7 GAL7 Epidermal LC Very highly expressed lectin, galactoside-binding, soluble, 9 LGALS9 HUAT DDC Exclusive nonexpression lectin, galactoside-binding, soluble, 10 LGALS10 CLC Blood pDC/MDDC High expression lectin, galactoside-binding, soluble, 12 LGALS12 GAL12, GRIP1 CD14+ mono Exclusive expression C-type lectin domain family 1, member A CLEC1A Blood pDC/epidermal LC Exclusive nonexpression C-type lectin domain family 2, member B CLEC2B AICL MDDC Exclusive nonexpression C-type lectin domain family 3, member B CLEC3B TN DDC/MDDC Exclusive expression C-type lectin domain family 4, member C CLEC4C BDCA2, CD303 Blood pDC Exclusive high expression C-type lectin domain family 4, member E CLEC4E MINCLE DDC Exclusive high expression C-type lectin domain family 4, member G CLEC4G LSECtin CD14+ DDC/MDDC Exclusive expression C-type lectin domain family 4, member K CLEC4K langerin, CD207 Epidermal LC Exclusive high expression C-type lectin domain family 4, member L CLEC4L DCSIGN, CD209 CD14+ DDC/MDDC Exclusive expression C-type lectin domain family 4, member M CLEC4M LSIGN, CD299 CD1a+ DDC Highly expressed C-type lectin domain family 5, member A CLEC5A MDL1 CD1a+ DDC/epidermal LC Exclusive expression

C-type lectin domain family 6, member A CLEC6A DECTIN-2, CLEC4N Blood pDC Exclusive nonexpression Downloaded from C-type lectin domain family 7, member A CLEC7A DECTIN-1, BGR Blood pDC Exclusive nonexpression C-type lectin domain family 8, member A CLEC8A OLR1, LOX1 Skin DC Exclusive expression C-type lectin domain family 12, member A CLEC12A MICL, DCAL-2, CLL1 CD1A+ DDC Exclusive nonexpression C-type lectin domain family 13, member B CLEC13B DEC205, LY75, CD205 DDC Nonexpression C-type lectin domain family 13, member D CLEC13D MRC1, CD206 DDC/MDDC Exclusive expression C-type lectin domain family 13, member D-like CLEC13DLa MRC1L1 DDC/MDDC Exclusive expression C-type lectin domain family 14, member A CLEC14A EGFR-5, CEG1 CD14+ DDC/MDDC Exclusive expression selectin L SELL CD62L, LAM1 Blood cells Exclusive expression http://www.jimmunol.org/ selectin P SELP CD62 CD1A+ DDC Exclusive nonexpression collectin 7 COLEC7 SFTPD Blood pDC Exclusive nonexpression collectin 12 COLEC12 CLP1 DDC/MDDC Very high expression TLR 2 TLR2 CD282 Blood pDC Exclusive nonexpression TLR 3 TLR3 CD283 Blood pDC Exclusive nonexpression TLR 7 TLR7 Blood pDCs Very high expression TLR 9 TLR9 CD289 Blood pDCs Very high expression solute carrier family 11 (proton-coupled SLC11A1 LSH, NRAMP CD14+/CD16+ mono Exclusive expression divalent metal ion transporters), member 1 + SWI/SNF-related, matrix-associated, SMARCD3 BAF60C CD14 mono Highly expressed by guest on September 26, 2021 actin-dependent regulator of chromatin, subfamily d, member 3 solute carrier family 5 SLC5A10 SGLT5 CD16+ mono Exclusive expression (sodium/glucose cotransporter), member 10 LY6/PLAUR domain containing 2 LYPD2 LYPDC2 CD16+ mono Highly expressed protein tyrosine phosphatase, PTPN3 PTPH1 CD16+ mono Highly expressed nonreceptor type 3 inverted formin, FH2 and WH2 domain INF2 CD16+ mono Exclusive nonexpression containing serine/threonine kinase 11 STK11 LKB1, PJS mDC Exclusive nonexpression Src-like adaptor 2 SLA2 SLAP2, MARS CD1a+ DDC Exclusive expression receptor accessory protein 1 REEP1 CD14+ DDC Exclusive expression plexin C1 PLXNC1 CD232, VESPR CD14+ DDC/MDDC Exclusive nonexpression ephrin-B3 EFNB3 EFL6, EPLG8, LERK8 Epidermal LC Exclusive expression protein kinase N1 PKN PAK1, PRK1 Epidermal LC Exclusive nonexpression chemokine (C-C motif) ligand 18 CCL18 AMAC1 MDDC Exclusive expression troponin 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. the skin, the CD1a+ DDCs and LCs clustered most closely, con- cursors. However, we did not observe a conversion of CD14+ sistent with the common expression of CD1a on their surface, with to CD1a+ cells after purification, indicating that a factor within the CD14+ DDCs forming a separate cluster. When we investi- the microenvironment of the skin is necessary to drive the conver- gated the effects of skin explant culture on isolated DDCs in situ, sion. Our findings in this study of expression profiles of segregated we saw a phenotypic shift from CD14-expressing cells to CD1a- populations based on anatomical location (blood versus skin) are expressing cells, with some cells expressing both markers (Fig. consistent with a recent murine DC microarray study that looked 3C, 3D). In addition, we found that after overnight culture, the at lymphoid resident DCs (39). proportion of CD1a+ cells isolated increased and CD14+ cells Recently, langerin-expressing CD103+ DDCs have been dis- decreased. These observations of close clustering of skin DCs and covered in mice (31). Langerin-expressing DDCs have also been a phenotypic shift after culture suggest that the dermal skin pop- reported in human skin; however, these have been considered as ulations may be related and interconvert from CD14+ to CD1a+ epidermal LCs in transit through the dermis (40), and there re- DDCs without direct origin from separate blood monocytic pre- mains speculation as to the existence of an equivalent population 76 LINEAGE RELATIONSHIP IN HUMAN DENDRITIC CELLS Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

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). in humans. Although we found that LCs were the only cell pop- possibility of minor contamination between CD1a-expressing LCs ulation that expressed high langerin levels, we did find that CD1a+ and DDCs. In addition, the principle components analysis showed DDCs (but not CD14+ DDCs) also expressed langerin,albeit an overlap between the CD1a+ DDC and LC populations, which at very low levels (Fig. 4C). We cannot completely exclude the also clustered closely together in the Illumina expression analysis. The Journal of Immunology 77

This suggests that a human langerin-expressing DDC subset may was exclusively not expressed by CD16+ monocyte. INF2 is in- exist within the CD1a+ DDC population but represent only a trace volved in immunologic synapse formation (48), and its non- population in resting skin. expression is consistent with limited ability of CD16+ monocytes to The differentiation of CD14+ monocytes into MDDCs using IL- form immunological synapses compared with DCs or other cells 4 and GM-CSF (19, 20) has provided a method for generating that form such synapses. 4) CD11c+ blood myeloid DCs did not large numbers of DCs for immunotherapy and a model for the exclusively express any genes. However, they alone did not express study of DC biology in vitro. Similarly, LC-like cells can be the serine/threonine kinase STK11. 5) pDCs showed the most generated in vitro either from CD34+ bone marrow or blood marked differences in their surface marker expression compared precursors cultured in GM-CSF and TNF-a (41), or from CD14+ with other ex vivo cells as expected. They exclusively expressed monocytes using TGF-b, GM-CSF, and IL-4 (42). Although hu- very high levels of CLEC4C (BDCA2), LGALS10 (CLC), TLR7,and man monocytes have been shown to differentiate into both mac- TLR9. In addition, they were the only cells not to express COLEC7, rophages and DCs after transendothelial migration (13), and a TLR2, TLR3,aswellasCLEC6A (dectin 2) CLEC7A (dectin 1), TGF-b–dependent pathway from CD14+ monocytes to LCs may which play a role in innate recognition of fungi (49). exist in vivo (12), it is unclear what the in vivo equivalents of these Skin cell populations uniquely express the low-density lipo- model DCs are. MDDCs have been proposed to most closely re- protein scavenger receptor CLEC8A (OLR1). Selectin L was not semble CD14+ DDCs partly because both express the CLR DC- expressed by skin DCs, consistent with an essential role in blood SIGN (25). leukocyte emigration through vascular endothelium (50). Presence LC-like cells derived from CD34+ or CD14+ precursors are or absence of some markers were speciﬁc for skin DC subpop- assumed to mimic LCs because of their high langerin expression, ulations. 1) Epidermal LCs uniquely express very high levels of Downloaded from but unlike LCs, they also express the CLRs DC-SIGN and MR, LGALS7 and langerin, as well as EFNB3, as previously shown by similar to MDDCs. In addition to expressing high levels of lan- others (51). This is consistent with known expression of LGALS7 gerin, MUTZ3 LCs lack the expression of DC-SIGN and MR (21), by epithelial cells (52). LGALS2 was expressed at much lower and for this reason, we chose to investigate this model LC. In levels than other cells. We found that our uncultured LCs did not support of their use as model skin DCs, both the MDDC and express TLR4 as previously reported by others (53), and expres-

MUTZ3 LC cell populations did reside within the general skin DC sion of TLR2 was detected in only two of the four LC donors. This http://www.jimmunol.org/ cluster (Fig. 2C). Consistent with their proposed similarity to is consistent with previous work (54), although a recent publica- CD14+ DDCs, the immature MDDCs clustered most closely to tion shows TLR2 and TLR4 expression on LCs after culture (55). this ex vivo cell type. However, the MUTZ3 LCs cells did not LCs did not express TLR5 or TLR9 and uniquely lacked PKN, cluster closely to LCs and rather formed the most discrete cluster which regulates NF-kB signaling (integral to TLR-mediated sig- within the skin cells alongside mature MDDCs, indicating that nal transduction) via phosphorylation of TRAF1 (56). 2) DDCs caution should be applied to findings derived from this model uniquely express LGALS1, which plays a role in leukocyte mi- system. However, they would remain a useful model for studies gration, modulates proinflammatory cytokine release (57), and involving surface receptors. mediates DC anergy (58), and CLEC4E (MINCLE), which plays In support of MDDCs as model DDCs, we found common patterns a role in recognizing pathogenic fungus (59). LGALS9, which by guest on September 26, 2021 of exclusive CLR expression, both uniquely expressing CLEC13D plays a pivotal role in T cell immunity (60), was uniquely not (mannose receptor), CLEC13DL, CLEC3B (TNA), and COLEC12. expressed by DDCs. 3) CD1a+ DDCs uniquely express SLA2, Furthermore, in support of a closer similarity to CD14+ DDCs than which has recently been shown to regulate DC maturation (61). In CD1a+ DDCs, we confirmed the finding that CLEC4L (DCSIGN) addition, they expressed much higher levels of CLEC4M (LSIGN) expression was restricted to CD14+ DDCs and MDDCs, and we shown to be associated with HIV transmission (62). CD1a+ DDCs also show that the related CLEC4G (LSECtin)andCLEC14A were were the only cell population that consistently did not express also uniquely expressed by these two cell types. Like DC-SIGN, the monocytic cell marker CELC12A (DCAL2). 4) CD14+ DDCs LSECtin is involved in pathogen binding and recognizes endoge- exclusively expressed REEP1, which is involved in targeting nous activated T cells (43), and CLEC14A has recently been iden- receptors to lipid rafts (63). Conversely, PLXNC1 was exclusively tified as an endothelial marker involved in cell migration (44). No nonexpressed. In murine DCs, PLXNC1 signaling inhibits integ- CLRs were uniquely expressed or nonexpressed by MDDCs and rins resulting in decreased cell adhesion and migration, and DCs LCs or CD1a+ DDCs. in mice lacking PLXNC1 showed a reduced capacity to stimulate In this study, we have identified additional specific markers for the T cells (64). different DC/monocyte subpopulations (Table II), which may serve The ontogeny of DCs, and their migration to and population of as a basis to more easily distinguish between subpopulations: 1) tissue have important implications for dissemination of infectious CD14+ and CD16+ blood monocytes specifically express SLC11A1, organisms. For example, HIV can readily infect or be carried by SLC5A10,andSMARCD3. SLC11A1 and SLC5A10 are both solute blood DCs or CD16+ monocytes (26), which marginalize (5) and carrier proteins. SLC5A10 has been shown to be exclusively ex- emigrate under inflammatory conditions to generate tissue DCs. pressed in myeloid lineage cells and plays a role in macrophage We have previously shown that different CLRs may bind HIV in differentiation where the recruitment of SWI/SNF complex is re- tissue (25), and recent work has suggested that within the skin, quired for its transcriptional activation (45). SMARCD3 is a core langerin-mediated binding by HIV may have differing effects component of the nuclear SWI/SNF complex linking the cell-type– depending on the state of the cell (65). In this study, we have specific function of the proteins encoded by these two genes. This now extended our understanding of DC–HIV interactions by complex is also required for HIV TAT-driven transcriptional acti- providing profiles for expression of surface markers on DC sub- vation (46). 2) CD14+ monocytes uniquely express LGALS12, sets in blood and skin that may be important in HIV entry and previously been shown to be involved in regulating the cell cycle virus carriage by these DC subsets. Such data are also relevant to (47). 3) CD16+ monocytes uniquely express LYPD2 and PTPN. No other epitheliotropic viruses such as HSV. Understanding these studies have been published on LYPD2, but PTPN3 is a member of processes can only be achieved by a study of primary cells and use the protein tyrosine phosphatase family that regulates a variety of of uniquely expressed and nonexpressed markers as we have iden- cellular processes including cell growth and differentiation. INF2 tified in this study. 78 LINEAGE RELATIONSHIP IN HUMAN DENDRITIC CELLS

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