Identification of Lineage Relationships and Novel Markers of Blood And
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Published November 26, 2012, doi:10.4049/jimmunol.1200779 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 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. 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 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 2 LINEAGE RELATIONSHIP IN HUMAN DENDRITIC CELLS 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 be- tween the human CD11c+ CD1c+ blood myeloid DCs as precursors Isolation of blood mononuclear cell populations and DCs was done as pre- viously 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 models, the development of committed DC precursors in blood isolated over Ficoll