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1.  The XC chemokine receptor 1 is a conserved selective marker of mammalian cells homologous to mouse CD8α+ dendritic cells 
The Journal of Experimental Medicine  2010;207(6):1283-1292.
Human BDCA3+ dendritic cells (DCs) were suggested to be homologous to mouse CD8α+ DCs. We demonstrate that human BDCA3+ DCs are more efficient than their BDCA1+ counterparts or plasmacytoid DCs (pDCs) in cross-presenting antigen and activating CD8+ T cells, which is similar to mouse CD8α+ DCs as compared with CD11b+ DCs or pDCs, although with more moderate differences between human DC subsets. Yet, no specific marker was known to be shared between homologous DC subsets across species. We found that XC chemokine receptor 1 (XCR1) is specifically expressed and active in mouse CD8α+, human BDCA3+, and sheep CD26+ DCs and is conserved across species. The mRNA encoding the XCR1 ligand chemokine (C motif) ligand 1 (XCL1) is selectively expressed in natural killer (NK) and CD8+ T lymphocytes at steady-state and is enhanced upon activation. Moreover, the Xcl1 mRNA is selectively expressed at high levels in central memory compared with naive CD8+ T lymphocytes. Finally, XCR1−/− mice have decreased early CD8+ T cell responses to Listeria monocytogenes infection, which is associated with higher bacterial loads early in infection. Therefore, XCR1 constitutes the first conserved specific marker for cell subsets homologous to mouse CD8α+ DCs in higher vertebrates and promotes their ability to activate early CD8+ T cell defenses against an intracellular pathogenic bacteria.
doi:10.1084/jem.20100223
PMCID: PMC2882835  PMID: 20479118
2.  TLR3 Signaling Promotes the Induction of Unique Human BDCA-3 Dendritic Cell Populations 
Conventional and plasmacytoid dendritic cells (cDCs and pDCs) are the two populations of DCs that can be readily identified in human blood. Conventional DCs have been subdivided into CD1c+, or blood dendritic cells antigen (BDCA) 1 and CD141+, or BDCA-3, DCs, each having both unique gene expression profiles and functions. BDCA-3 DCs express high levels of toll-like receptor 3 and upon stimulation with Poly I:C secrete IFN-β, CXCL10, and IL-12p70. In this article, we show that activation of human BDCA-3 DCs with Poly I:C induces the expression of activation markers (CD40, CD80, and CD86) and immunoglobulin-like transcript (ILT) 3 and 4. This Poly I:C stimulation results in four populations identifiable by flow cytometry based on their expression of ILT3 and ILT4. We focused our efforts on profiling the ILT4− and ILT4+ DCs. These ILT-expressing BDCA-3 populations exhibit similar levels of activation as measured by CD40, CD80, and CD86; however, they exhibit differential cytokine secretion profiles, unique gene signatures, and vary in their ability to prime allogenic naïve T cells. Taken together, these data illustrate that within a pool of BDCA-3 DCs, there are cells poised to respond differently to a given input stimulus with unique output of immune functions.
doi:10.3389/fimmu.2016.00088
PMCID: PMC4789364  PMID: 27014268
dendritic cell; TLR3; ILT3; ILT4; BDCA-3
3.  Characterization of species-specific genes regulated by E2-2 in human plasmacytoid dendritic cells 
Scientific Reports  2015;5:10752.
Dendritic cells (DCs) are sentinels of the immune system and comprise two distinct subsets: conventional DCs (cDCs) and plasmacytoid DCs (pDCs). Human pDCs are distinguished from mouse pDCs phenotypically and functionally. Basic helix-loop-helix protein E2-2 is defined as an essential transcription factor for mouse pDC development, cell fate maintenance and gene programe. It is unknown whether E2-2 regulation contributes to this species-specific difference. Here we investigated the function of E2-2 in human pDCs and screened human-specific genes regulated by E2-2. Reduced E2-2 expression in human pDC cell line GEN2.2 resulted in diminished IFN-α production in response to CpG but elevated antigen presentation capacity. Gene expression profiling showed that E2-2 silence down-regulated pDC signature genes but up-regulated cDC signature genes. Thirty human-specific genes regulated by E2-2 knockdown were identified. Among these genes, we confirmed that expression of Siglec-6 was inhibited by E2-2. Further more, Siglec-6 was expressed at a higher level on a human pDC subset with drastically lower expression of E2-2. Collectively, these results highlight that E2-2 modulates pDC function in a species-specific manner, which may provide insights for pDC development and functions.
doi:10.1038/srep10752
PMCID: PMC4505321  PMID: 26182859
4.  BDCA2/FcɛRIγ Complex Signals through a Novel BCR-Like Pathway in Human Plasmacytoid Dendritic Cells 
PLoS Biology  2007;5(10):e248.
Dendritic cells are equipped with lectin receptors to sense the extracellular environment and modulate cellular responses. Human plasmacytoid dendritic cells (pDCs) uniquely express blood dendritic cell antigen 2 (BDCA2) protein, a C-type lectin lacking an identifiable signaling motif. We demonstrate here that BDCA2 forms a complex with the transmembrane adapter FcɛRIγ. Through pathway analysis, we identified a comprehensive signaling machinery in human pDCs, similar to that which operates downstream of the B cell receptor (BCR), which is distinct from the system involved in T cell receptor (TCR) signaling. BDCA2 crosslinking resulted in the activation of the BCR-like cascade, which potently suppressed the ability of pDCs to produce type I interferon and other cytokines in response to Toll-like receptor ligands. Therefore, by associating with FcɛRIγ, BDCA2 activates a novel BCR-like signaling pathway to regulate the immune functions of pDCs.
Author Summary
Dendritic cells (DCs) are specialized sentinels in the immune system that detect invading pathogens and, upon activation, initiate immune responses. DCs express C-type lectin receptors on their surface, which facilitate antigen capture. A distinct population of DCs, called plasmacytoid DCs (pDCs), display an extraordinary ability to rapidly make huge amounts of antiviral interferon (IFN) against viral infections. Human pDCs uniquely express a C-type lectin named BDCA2 that potently regulates pDCs function, yet the mechanism of how BDCA2 transduces signals is unknown. We show here that BDCA2 forms a complex with the transmembrane adapter FcɛRIγ. Using signaling pathway analysis, we discovered a comprehensive signaling machinery in human pDCs, similar to that which operates downstream of B cell receptors (BCRs), but distinct from the pathway involved in T cell receptor signaling. By associating with FcɛRIγ, BDCA2 activates a novel BCR-like signaling pathway to regulate the immune functions of pDCs. Since several pDC receptors use this pathway to modulate IFN and cytokine responses, these findings will guide more studies on how pDCs are regulated. Such mechanisms may lead to potential therapeutic interventions in autoimmune diseases involving hyperactivated pDCs, such as systemic lupus erythematosus and psoriasis.
Plasmacytoid dendritic cells (pDCs) are renowned for their production of type 1 interferon in response to viral infection, which is signified by Toll-like receptor (TLR) activation. Here, blood dendritic cell antigen 2(BDCA2), a C-type lectin receptor expressed uniquely on pDCs, is shown to block the ultimate effectors of TLR signaling via a novel pathway.
doi:10.1371/journal.pbio.0050248
PMCID: PMC1971124  PMID: 17850179
5.  Proteomics of Human Dendritic Cell Subsets Reveals Subset-Specific Surface Markers and Differential Inflammasome Function 
Cell Reports  2016;16(11):2953-2966.
Summary
Dendritic cells (DCs) play a key role in orchestrating adaptive immune responses. In human blood, three distinct subsets exist: plasmacytoid DCs (pDCs) and BDCA3+ and CD1c+ myeloid DCs. In addition, a DC-like CD16+ monocyte has been reported. Although RNA-expression profiles have been previously compared, protein expression data may provide a different picture. Here, we exploited label-free quantitative mass spectrometry to compare and identify differences in primary human DC subset proteins. Moreover, we integrated these proteomic data with existing mRNA data to derive robust cell-specific expression signatures with more than 400 differentially expressed proteins between subsets, forming a solid basis for investigation of subset-specific functions. We illustrated this by extracting subset identification markers and by demonstrating that pDCs lack caspase-1 and only express low levels of other inflammasome-related proteins. In accordance, pDCs were incapable of interleukin (IL)-1β secretion in response to ATP.
Graphical Abstract
Image 1
Highlights
•We present a comprehensive quantitative proteome comparison of primary human DC subsets•Proteome comparison reveals many expression differences between DC subsets•We provide a resource to derive markers and examine subset functional specialization•pDCs lack caspase-1 and have a decreased inflammasome response
Worah et al. present a comprehensive quantitative proteomic comparison of four human blood-derived DC-like subsets. Through integration of proteomic and transcriptomic data, the authors derive expression signatures for each subset that provide a resource for study of subset functional specialization.
doi:10.1016/j.celrep.2016.08.023
PMCID: PMC5039226  PMID: 27626665
6.  Profiling of primary peripheral blood- and monocyte-derived dendritic cells using monoclonal antibodies from the HLDA10 Workshop in Wollongong, Australia 
Dendritic cells (DCs) arise from hematopoietic stem cells and develop into a discrete cellular lineage distinct from other leucocytes. Mainly three phenotypically and functionally distinct DC subsets are described in the human peripheral blood (PB): plasmacytoid DCs (pDCs), which express the key marker CD303 (BDCA-2), and two myeloid DC subsets (CD1c+ DC (mDC1) and CD141+ DC (mDC2)), which express the key markers CD1c (BDCA-1) and CD141 (BDCA-3), respectively. In addition to these primary cell subsets, DCs can also be generated in vitro from either CD34+ stem/progenitor cells in the presence of Flt3 (Fms-related tyrosine kinase 3) ligand or from CD14+ monocytes (monocyte-derived DCs (mo-DCs)) in the presence of granulocyte–macrophage colony-stimulating factor+interleukin-4 (GM-CSF+IL-4). Here we compare the reactivity patterns of HLDA10 antibodies (monoclonal antibody (mAb)) with pDCs, CD1c+ DCs and CD141+ DCs, as well as with CD14+-derived mo-DCs cultured for 7 days in the presence of 100 ng/ml GM-CSF plus 20 ng/ml IL-4. A detailed profiling of these DC subsets based on immunophenotyping and multicolour flow cytometry analysis is presented. Using the panel of HLDA10 Workshop mAb, we could verify known targets selectively expressed on discrete DC subsets including CD370 as a selective marker for CD141+ DCs and CD366 as a marker for both myeloid subsets. In addition, vimentin and other markers are heterogeneously expressed on all three subsets, suggesting the existence of so far not identified DC subsets.
doi:10.1038/cti.2015.29
PMCID: PMC4673437  PMID: 26682057
7.  STING-Licensed Macrophages Prime Type I IFN Production by Plasmacytoid Dendritic Cells in the Bone Marrow during Severe Plasmodium yoelii Malaria 
PLoS Pathogens  2016;12(10):e1005975.
Malaria remains a global health burden causing significant morbidity, yet the mechanisms underlying disease outcomes and protection are poorly understood. Herein, we analyzed the peripheral blood of a unique cohort of Malawian children with severe malaria, and performed a comprehensive overview of blood leukocytes and inflammatory mediators present in these patients. We reveal robust immune cell activation, notably of CD14+ inflammatory monocytes, NK cells and plasmacytoid dendritic cells (pDCs) that is associated with very high inflammation. Using the Plasmodium yoelii 17X YM surrogate mouse model of lethal malaria, we report a comparable pattern of immune cell activation and inflammation and found that type I IFN represents a key checkpoint for disease outcomes. Compared to wild type mice, mice lacking the type I interferon (IFN) receptor exhibited a significant decrease in immune cell activation and inflammatory response, ultimately surviving the infection. We demonstrate that pDCs were the major producers of systemic type I IFN in the bone marrow and the blood of infected mice, via TLR7/MyD88-mediated recognition of Plasmodium parasites. This robust type I IFN production required priming of pDCs by CD169+ macrophages undergoing activation upon STING-mediated sensing of parasites in the bone marrow. pDCs and macrophages displayed prolonged interactions in this compartment in infected mice as visualized by intravital microscopy. Altogether our findings describe a novel mechanism of pDC activation in vivo and precise stepwise cell/cell interactions taking place during severe malaria that contribute to immune cell activation and inflammation, and subsequent disease outcomes.
Author Summary
The Plasmodium parasite is the number one killer among human parasitic diseases worldwide. Protection is associated with length of exposure for people living in endemic areas, with severe disease primarily affecting young children. Inflammation is a key component in the pathophysiology in malaria, and disease severity has been linked to the degree of activation of the immune system. However, the underlying mechanisms of protection and disease outcomes remain poorly understood. We provide a comprehensive analysis of peripheral blood immune cells obtained from a cohort of children with severe malaria. Our results show heightened inflammation and immune cell activation, in particular for monocytes, natural killer cells, and plasmacytoid dendritic cells (pDCs). We have also utilized a mouse model of lethal malaria that recapitulates many features identified in this cohort of severe malaria patients to examine drivers of immune cell activation and inflammation. Our studies provide evidence that type I interferon (IFN) acts as an early switch in inducing a potent inflammatory response in the infected host. Type I IFN production is massively produced in the bone marrow and the blood of infected mice by plasmacytoid dendritic cells (pDCs), a subset of DCs. We also demonstrate that resident macrophages in the bone marrow, control type I IFN production by the pDCs. We define how both myeloid cells “sense” the parasite to initiate the host immune response and report a previously uncharacterized physical interaction between pDCs and macrophages in the bone marrow as visualized by intravital microscopy in vivo. Our results define cellular processes underlying the marked inflammation of severe malaria and could open novel therapeutic opportunities to improve outcomes in this important human infectious disease.
doi:10.1371/journal.ppat.1005975
PMCID: PMC5085251  PMID: 27792766
8.  Evaluation of the BDCA2-DTR Transgenic Mouse Model in Chronic and Acute Inflammation 
PLoS ONE  2015;10(8):e0134176.
Background and Aims
Plasmacytoid dendritic cells (pDCs) are a small subset of dendritic cells and the main producers of type I interferons. Besides their contribution to tolerance, they are known to be involved in autoimmune diseases and have recently been implicated in atherosclerosis. However, their precise involvement, particularly in advanced lesion development, remains elusive. Hence, we investigated the role of pDCs in atherogenesis vs atheroprogression by specifically depleting this cell population using the BDCA2-DTR mouse model bred to Apolipoprotein E (Apoe-/-) deficient mice.
Methods and Results
Our results revealed that continuous diphtheria toxin-induced pDC depletion in Apoe-/- BDCA2-DTR mice receiving a high-fat diet (HFD) for 4 weeks did not alter lesion size or composition. Instead, these mice displayed increased B cell numbers and altered levels of inflammatory cytokines. Analysis of depletion efficiency showed that complete pDC depletion could only be sustained for one week and reoccurring pDCs sorted after 4 weeks did not express DTR anymore. Consequently, we analyzed lesion development in a model of partial carotid ligation, inducing established lesions after 5 weeks of HFD feeding, and only depleted pDCs during the last week of 5 weeks HFD feeding. Despite short-term, but efficient pDC depletion, we observed no differences in atherosclerotic lesion development, but changes in inflammatory cytokine titers. To assure the functionality of the BDCA2-DTR model in acute settings, we additionally examined the effect of pDC depletion in an indirect acute lung injury (iALI) model. This time, efficient pDC depletion resulted in a significantly reduced macrophage and neutrophil accumulation in the lung 12 hours after LPS challenge, underlining a pro-inflammatory role of pDCs in the innate immune response in iALI.
Conclusion
Taken together, the BDCA2-DTR mouse model only allows efficient pDC depletion for one week, which subsequently restricts its usability to more acute but not chronic inflammatory disease models.
doi:10.1371/journal.pone.0134176
PMCID: PMC4529211  PMID: 26252890
9.  Rotavirus Structural Proteins and dsRNA Are Required for the Human Primary Plasmacytoid Dendritic Cell IFNα Response 
PLoS Pathogens  2010;6(6):e1000931.
Rotaviruses are the leading cause of severe dehydrating diarrhea in children worldwide. Rotavirus-induced immune responses, especially the T and B cell responses, have been extensively characterized; however, little is known about innate immune mechanisms involved in the control of rotavirus infection. Although increased levels of systemic type I interferon (IFNα and β) correlate with accelerated resolution of rotavirus disease, multiple rotavirus strains, including rhesus rotavirus (RRV), have been demonstrated to antagonize type I IFN production in a variety of epithelial and fibroblast cell types through several mechanisms, including degradation of multiple interferon regulatory factors by a viral nonstructural protein. This report demonstrates that stimulation of highly purified primary human peripheral plasmacytoid dendritic cells (pDCs) with either live or inactivated RRV induces substantial IFNα production by a subset of pDCs in which RRV does not replicate. Characterization of pDC responses to viral stimulus by flow cytometry and Luminex revealed that RRV replicates in a small subset of human primary pDCs and, in this RRV-permissive small subset, IFNα production is diminished. pDC activation and maturation were observed independently of viral replication and were enhanced in cells in which virus replicates. Production of IFNα by pDCs following RRV exposure required viral dsRNA and surface proteins, but neither viral replication nor activation by trypsin cleavage of VP4. These results demonstrate that a minor subset of purified primary human peripheral pDCs are permissive to RRV infection, and that pDCs retain functionality following RRV stimulus. Additionally, this study demonstrates trypsin-independent infection of primary peripheral cells by rotavirus, which may allow for the establishment of extraintestinal viremia and antigenemia. Importantly, these data provide the first evidence of IFNα induction in primary human pDCs by a dsRNA virus, while simultaneously demonstrating impaired IFNα production in primary human cells in which RRV replicates. Rotavirus infection of primary human pDCs provides a powerful experimental system for the study of mechanisms underlying pDC-mediated innate immunity to viral infection and reveals a potentially novel dsRNA-dependent pathway of IFNα induction.
Author Summary
Rotaviruses cause severe dehydrating diarrhea and are a leading cause of death in children worldwide. A potent antiviral, interferon-α (IFNα), is rapidly secreted by plasmacytoid dendritic cells (pDCs) in response to viral single-stranded RNA or DNA genomes. Here, we examined the effects of rotavirus on pDCs purified from human blood. We found that very few pDCs supported rotavirus replication, and that pDCs retained similar functionality in response to live or inactivated rotaviruses. While pDCs produced large quantities of IFNα shortly after rotavirus exposure, this was impaired in cells supporting viral replication. We also found that two viral proteins and the rotavirus double-stranded RNA genome were required for the initiation of the pDC IFNα response to rotavirus. Additionally, we found that cleavage of one of these viral proteins, a traditional prerequisite for rotavirus infection in other cell types, was not required for the infection of pDCs or production of IFNα. This may enable the host to rapidly initiate an immune response to rotavirus that subsequently restricts infection to the intestine and contributes to the resolution of disease. Our study provides novel insight into the interaction between rotavirus and the host innate immune response, and also identifies a unique mechanism for the production of IFNα by pDCs.
doi:10.1371/journal.ppat.1000931
PMCID: PMC2880586  PMID: 20532161
10.  A Novel Mechanism for Binding of Galactose-terminated Glycans by the C-type Carbohydrate Recognition Domain in Blood Dendritic Cell Antigen 2* 
The Journal of Biological Chemistry  2015;290(27):16759-16771.
Background: BDCA-2 is an anti-inflammatory receptor uniquely expressed on plasmacytoid dendritic cells.
Results: Glycan arrays and x-ray structural analysis have been used to define the mechanism of sugar binding to BDCA-2.
Conclusion: BDCA-2 binds selectively to glycans containing the epitope Galβ1–3/4GlcNAcβ1–2Man.
Significance: Binding of BDCA-2 to unusual galactose-terminated glycans on IgG or other serum glycoproteins provides a potential mechanism for modulating the immune response.
Blood dendritic cell antigen 2 (BDCA-2; also designated CLEC4C or CD303) is uniquely expressed on plasmacytoid dendritic cells. Stimulation of BDCA-2 with antibodies leads to an anti-inflammatory response in these cells, but the natural ligands for the receptor are not known. The C-type carbohydrate recognition domain in the extracellular portion of BDCA-2 contains a signature motif typical of C-type animal lectins that bind mannose, glucose, or GlcNAc, yet it has been reported that BDCA-2 binds selectively to galactose-terminated, biantennary N-linked glycans. A combination of glycan array analysis and binding competition studies with monosaccharides and natural and synthetic oligosaccharides have been used to define the binding epitope for BDCA-2 as the trisaccharide Galβ1–3/4GlcNAcβ1–2Man. X-ray crystallography and mutagenesis studies show that mannose is ligated to the conserved Ca2+ in the primary binding site that is characteristic of C-type carbohydrate recognition domains, and the GlcNAc and galactose residues make additional interactions in a wide, shallow groove adjacent to the primary binding site. As predicted from these studies, BDCA-2 binds to IgG, which bears galactose-terminated glycans that are not commonly found attached to other serum glycoproteins. Thus, BDCA-2 has the potential to serve as a previously unrecognized immunoglobulin Fc receptor.
doi:10.1074/jbc.M115.660613
PMCID: PMC4505424  PMID: 25995448
carbohydrate-binding protein; crystal structure; glycobiology; glycoprotein; lectin; CD303; CLEC4C
11.  Human BDCA2+CD123+CD56+ dendritic cells (DCs) related to blastic plasmacytoid dendritic cell neoplasm represent a unique myeloid DC subset 
Protein & Cell  2015;6(4):297-306.
ABSTRACT
Dendritic cells (DCs) comprise two functionally distinct subsets: plasmacytoid DCs (pDCs) and myeloid DCs (mDCs). pDCs are specialized in rapid and massive secretion of type I interferon (IFN-I) in response to nucleic acids through Toll like receptor (TLR)-7 or TLR-9. In this report, we characterized a CD56+ DC population that express typical pDC markers including CD123 and BDCA2 but produce much less IFN-I comparing with pDCs. In addition, CD56+ DCs cluster together with mDCs but not pDCs by genome-wide transcriptional profiling. Accordingly, CD56+ DCs functionally resemble mDCs by producing IL-12 upon TLR4 stimulation and priming naïve T cells without prior activation. These data suggest that the CD56+ DCs represent a novel mDC subset mixed with some pDC features. A CD4+CD56+ hematological malignancy was classified as blastic plasmacytoid dendritic cell neoplasm (BPDCN) due to its expression of characteristic molecules of pDCs. However, we demonstrated that BPDCN is closer to CD56+ DCs than pDCs by global gene-expression profiling. Thus, we propose that the CD4+CD56+ neoplasm may be a tumor counterpart of CD56+ mDCs but not pDCs.
Electronic supplementary material
The online version of this article (doi:10.1007/s13238-015-0140-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s13238-015-0140-x
PMCID: PMC4383756  PMID: 25779340
dendritic cells; CD56+ DC; pDC; mDC; BPDCN
12.  Human BDCA2+CD123+CD56+ dendritic cells (DCs) related to blastic plasmacytoid dendritic cell neoplasm represent a unique myeloid DC subset 
Protein & Cell  2015;6(4):297-306.
Dendritic cells (DCs) comprise two functionally distinct subsets: plasmacytoid DCs (pDCs) and myeloid DCs (mDCs). pDCs are specialized in rapid and massive secretion of type I interferon (IFN-I) in response to nucleic acids through Toll like receptor (TLR)-7 or TLR-9. In this report, we characterized a CD56+ DC population that express typical pDC markers including CD123 and BDCA2 but produce much less IFN-I comparing with pDCs. In addition, CD56+ DCs cluster together with mDCs but not pDCs by genome-wide transcriptional profiling. Accordingly, CD56+ DCs functionally resemble mDCs by producing IL-12 upon TLR4 stimulation and priming naïve T cells without prior activation. These data suggest that the CD56+ DCs represent a novel mDC subset mixed with some pDC features. A CD4+CD56+ hematological malignancy was classified as blastic plasmacytoid dendritic cell neoplasm (BPDCN) due to its expression of characteristic molecules of pDCs. However, we demonstrated that BPDCN is closer to CD56+ DCs than pDCs by global gene-expression profiling. Thus, we propose that the CD4+CD56+ neoplasm may be a tumor counterpart of CD56+ mDCs but not pDCs.
Electronic supplementary material
The online version of this article (doi:10.1007/s13238-015-0140-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s13238-015-0140-x
PMCID: PMC4383756  PMID: 25779340
dendritic cells; CD56+ DC; pDC; mDC; BPDCN
13.  Modular expression analysis reveals functional conservation between human Langerhans cells and mouse cross-priming dendritic cells 
In depth phenotyping and functional analysis of skin dendritic cell subsets suggests that the function of Langerhans cells may not be conserved between mouse and human and supports the idea that human Langernhans cells may be a relevant therapeutic target.
Characterization of functionally distinct dendritic cell (DC) subsets in mice has fueled interest in whether analogous counterparts exist in humans. Transcriptional modules of coordinately expressed genes were used for defining shared functions between the species. Comparing modules derived from four human skin DC subsets and modules derived from the Immunological Genome Project database for all mouse DC subsets revealed that human Langerhans cells (LCs) and the mouse XCR1+CD8α+CD103+ DCs shared the class I–mediated antigen processing and cross-presentation transcriptional modules that were not seen in mouse LCs. Furthermore, human LCs were enriched in a transcriptional signature specific to the blood cross-presenting CD141/BDCA-3+ DCs, the proposed equivalent to mouse CD8α+ DCs. Consistent with our analysis, LCs were highly adept at inducing primary CTL responses. Thus, our study suggests that the function of LCs may not be conserved between mouse and human and supports human LCs as an especially relevant therapeutic target.
doi:10.1084/jem.20131675
PMCID: PMC4419344  PMID: 25918340
14.  Immunity to Pathogens Taught by Specialized Human Dendritic Cell Subsets 
Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) that have a key role in immune responses because they bridge the innate and adaptive arms of the immune system. They mature upon recognition of pathogens and upregulate MHC molecules and costimulatory receptors to activate antigen-specific CD4+ and CD8+ T cells. It is now well established that DCs are not a homogeneous population but are composed of different subsets with specialized functions in immune responses to specific pathogens. Upon viral infections, plasmacytoid DCs (pDCs) rapidly produce large amounts of IFN-α, which has potent antiviral functions and activates several other immune cells. However, pDCs are not particularly potent APCs and induce the tolerogenic cytokine IL-10 in CD4+ T cells. In contrast, myeloid DCs (mDCs) are very potent APCs and possess the unique capacity to prime naive T cells and consequently to initiate a primary adaptive immune response. Different subsets of mDCs with specialized functions have been identified. In mice, CD8α+ mDCs capture antigenic material from necrotic cells, secrete high levels of IL-12, and prime Th1 and cytotoxic T-cell responses to control intracellular pathogens. Conversely, CD8α− mDCs preferentially prime CD4+ T cells and promote Th2 or Th17 differentiation. BDCA-3+ mDC2 are the human homologue of CD8α+ mDCs, since they share the expression of several key molecules, the capacity to cross-present antigens to CD8+ T-cells and to produce IFN-λ. However, although several features of the DC network are conserved between humans and mice, the expression of several toll-like receptors as well as the production of cytokines that regulate T-cell differentiation are different. Intriguingly, recent data suggest specific roles for human DC subsets in immune responses against individual pathogens. The biology of human DC subsets holds the promise to be exploitable in translational medicine, in particular for the development of vaccines against persistent infections or cancer.
doi:10.3389/fimmu.2015.00527
PMCID: PMC4603245  PMID: 26528289
dendritic cells; cytokines; toll-like receptors; T-cell differentiation; cytotoxic T cells
15.  Myeloid derived suppressor and dendritic cell subsets are related to clinical outcome in prostate cancer patients treated with prostate GVAX and ipilimumab 
Background
Cancer-related disturbances in myeloid lineage development, marked by high levels of myeloid-derived suppressor cells (MDSC) and impaired dendritic cell (DC) development, are associated with poor clinical outcome due to immune escape and therapy resistance. Redressing this balance may therefore be of clinical benefit. Here we investigated the effects of combined Prostate GVAX/ipilimumab immunotherapy on myeloid subsets in peripheral blood of castration-resistant prostate cancer (CRPC) patients as well as the putative predictive value of baseline and on-treatment myeloid parameters on clinical outcome.
Methods
Patients with CRPC (n = 28) received thirteen intradermal administrations of Prostate GVAX, consisting of two allogeneic GM-CSF-transduced and irradiated prostate cancer cell lines (LN-CaP and PC3) and six infusions of escalating doses of anti-CTLA4/ipilimumab. Frequencies and activation status of peripheral blood DC (PBDC) and MDSC were determined before, during and after treatment by flowcytometric analysis and related to clinical benefit.
Results
Significant treatment-induced activation of conventional and plasmacytoid DC subsets (cDC and pDC) was observed, which in the case of BDCA1/CD1c+ cDC1 and MDC8+/6-sulfoLacNAc+ inflammatory cDC3 was associated with significantly prolonged overall survival (OS), but also with the development of autoimmune-related adverse events. High pre-treatment levels of CD14+HLA-DR−monocytic MDSC (mMDSC) were associated with reduced OS. Unsupervised clustering of these myeloid biomarkers revealed particular survival advantage in a group of patients with high treatment-induced PBDC activation and low pretreatment frequencies of suppressive mMDSC in conjunction with our previously identified lymphoid biomarker of high pretreatment CD4+CTLA4+ T cell frequencies.
Conclusions
Our data demonstrate that DC and MDSC subsets are affected by prostate GVAX/ipilimumab therapy and that myeloid profiling may contribute to the identification of patients with possible clinical benefit of Prostate GVAX/ipilimumab treatment.
Electronic supplementary material
The online version of this article (doi:10.1186/s40425-014-0031-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s40425-014-0031-3
PMCID: PMC4507359  PMID: 26196012
Ipilimumab; Prostate GVAX; Biomarker; Patient selection; Survival prediction
16.  Dendritic Cell Subtypes from Lymph Nodes and Blood Show Contrasted Gene Expression Programs upon Bluetongue Virus Infection 
Journal of Virology  2013;87(16):9333-9343.
Human and animal hemorrhagic viruses initially target dendritic cells (DCs). It has been proposed, but not documented, that both plasmacytoid DCs (pDCs) and conventional DCs (cDCs) may participate in the cytokine storm encountered in these infections. In order to evaluate the contribution of DCs in hemorrhagic virus pathogenesis, we performed a genome-wide expression analysis during infection by Bluetongue virus (BTV), a double-stranded RNA virus that induces hemorrhagic fever in sheep and initially infects cDCs. Both pDCs and cDCs accumulated in regional lymph nodes and spleen during BTV infection. The gene response profiles were performed at the onset of the disease and markedly differed with the DC subtypes and their lymphoid organ location. An integrative knowledge-based analysis revealed that blood pDCs displayed a gene signature related to activation of systemic inflammation and permeability of vasculature. In contrast, the gene profile of pDCs and cDCs in lymph nodes was oriented to inhibition of inflammation, whereas spleen cDCs did not show a clear functional orientation. These analyses indicate that tissue location and DC subtype affect the functional gene expression program induced by BTV and suggest the involvement of blood pDCs in the inflammation and plasma leakage/hemorrhage during BTV infection in the real natural host of the virus. These findings open the avenue to target DCs for therapeutic interventions in viral hemorrhagic diseases.
doi:10.1128/JVI.00631-13
PMCID: PMC3754054  PMID: 23785206
17.  Crosstalk between human DC subsets promotes antibacterial activity and CD8+ T-cell stimulation in response to bacille Calmette-Guérin 
European Journal of Immunology  2013;44(1):80-92.
To date, little is known about the unique contributions of specialized human DC subsets to protection against tuberculosis (TB). Here, we focus on the role of human plasmacytoid (p)DCs and myeloid (m)DCs in the immune response to the TB vaccine bacille Calmette-Guérin (BCG). Ex vivo DC subsets from human peripheral blood were purified and infected with BCG expressing GFP to distinguish between infected and noninfected cells. BDCA-1+ myeloid DCs were more susceptible than BDCA-3+ mDCs to BCG infection. Plasmacytoid DCs have poor phagocytic activity but are equipped with endocytic receptors and can be activated by bystander stimulation. Consequently, the mutual interaction of the two DC subsets in response to BCG was analyzed. We found that pDCs were activated by BCG-infected BDCA-1+ mDCs to upregulate maturation markers and to produce granzyme B, but not IFN-α. Reciprocally, the presence of activated pDCs enhanced mycobacterial growth control by infected mDCs and increased IL-1β availability. The synergy between the two DC subsets promoted BCG-specific CD8+ T-cell stimulation and the role of BCG-infected BDCA-1+ mDCs could not be efficiently replaced by infected BDCA-3+ mDCs in the crosstalk with pDCs. We conclude that mDC–pDC crosstalk should be exploited for rational design of next-generation TB vaccines.
doi:10.1002/eji.201343797
PMCID: PMC3992850  PMID: 24114554
Bacille Calmette-Guérin (BCG); BDCA-1+ myeloid DCs; CD8+ T cells; human; plasmacytoid DCs; tuberculosis
18.  ETO family protein Mtg16 regulates the balance of dendritic cell subsets by repressing Id2 
The Journal of Experimental Medicine  2014;211(8):1623-1635.
Transcriptional cofactor of the ETO family Mtg16 promotes pDCs and restricts cDC differentiation in part by repressing Id2.
Dendritic cells (DCs) comprise two major subsets, the interferon (IFN)-producing plasmacytoid DCs (pDCs) and antigen-presenting classical DCs (cDCs). The development of pDCs is promoted by E protein transcription factor E2-2, whereas E protein antagonist Id2 is specifically absent from pDCs. Conversely, Id2 is prominently expressed in cDCs and promotes CD8+ cDC development. The mechanisms that control the balance between E and Id proteins during DC subset specification remain unknown. We found that the loss of Mtg16, a transcriptional cofactor of the ETO protein family, profoundly impaired pDC development and pDC-dependent IFN response. The residual Mtg16-deficient pDCs showed aberrant phenotype, including the expression of myeloid marker CD11b. Conversely, the development of cDC progenitors (pre-DCs) and of CD8+ cDCs was enhanced. Genome-wide expression and DNA-binding analysis identified Id2 as a direct target of Mtg16. Mtg16-deficient cDC progenitors and pDCs showed aberrant induction of Id2, and the deletion of Id2 facilitated the impaired development of Mtg16-deficient pDCs. Thus, Mtg16 promotes pDC differentiation and restricts cDC development in part by repressing Id2, revealing a cell-intrinsic mechanism that controls subset balance during DC development.
doi:10.1084/jem.20132121
PMCID: PMC4113936  PMID: 24980046
19.  Continuous expression of the transcription factor E2-2 maintains the cell fate of mature plasmacytoid dendritic cells 
Immunity  2010;33(6):905-916.
Summary
The interferon-producing plasmacytoid dendritic cells (pDCs) share common progenitors with antigen-presenting classical dendritic cells (cDCs), yet they possess distinct morphology and molecular features resembling those of lymphocytes. It is unclear whether the unique cell fate of pDCs is actively maintained in the steady state. We report that the deletion of transcription factor E2-2 from mature peripheral pDCs caused their spontaneous differentiation into cells with cDC properties. This included the loss of pDC markers, increase in MHC class II expression and T cell priming capacity, acquisition of dendritic morphology and induction of cDC signature genes. Genome-wide chromatin immunoprecipitation revealed direct binding of E2-2 to key pDC-specific and lymphoid genes, as well as to certain genes enriched in cDCs. Thus, E2-2 actively maintains the cell fate of mature pDCs and opposes the “default” cDC fate, in part through direct regulation of lineage-specific gene expression programs.
doi:10.1016/j.immuni.2010.11.023
PMCID: PMC3010277  PMID: 21145760
20.  Genetic profiles of plasmacytoid (BDCA-4 expressing) DC subtypes-clues to DC subtype function in vivo 
Among the dendritic cell (DC) subsets, plasmacytoid DC’s (pDC) are thought to be important in the generation of both antiviral and antitumor responses. While pDC may be useful in developing dendritic cell-based tumor vaccines, the low frequency of these cells in the peripheral blood has hampered attempts to understand their biology. To provide better insight into the biology of pDC, we isolated these unperturbed cells from the peripheral blood of healthy donors in order to further characterize their gene expression. Using gene array technology we compared the genetic profiles of these cells to those of CD14+ monocytes isolated from the same donors and found several immune related genes upregulated in this cell population. This is the first description, to our knowledge, of gene expression in this subset of DCs obtained from the peripheral blood of adult human donors without exposure in vitro to cytokine or growth factors. Understanding the natural genetic profiles of this dendritic cell subtype as well as others such as the BDCA-1 expressing myeloid DCs may enable us to manipulate these cells ex-vivo to generate enhanced DC-based tumor vaccines inducing more robust antitumor responses.
doi:10.1186/2162-3619-2-8
PMCID: PMC3608935  PMID: 23497451
Plasmacytoid dendritic cells; Gene expression; Granzyme B
21.  Hepatitis C Virus Stimulates Murine CD8α-Like Dendritic Cells to Produce Type I Interferon in a TRIF-Dependent Manner 
PLoS Pathogens  2016;12(7):e1005736.
Hepatitis C virus (HCV) induces interferon (IFN) stimulated genes in the liver despite of distinct innate immune evasion mechanisms, suggesting that beyond HCV infected cells other cell types contribute to innate immune activation. Upon coculture with HCV replicating cells, human CD141+ myeloid dendritic cells (DC) produce type III IFN, whereas plasmacytoid dendritic cells (pDC) mount type I IFN responses. Due to limitations in the genetic manipulation of primary human DCs, we explored HCV mediated stimulation of murine DC subsets. Coculture of HCV RNA transfected human or murine hepatoma cells with murine bone marrow-derived DC cultures revealed that only Flt3-L DC cultures, but not GM-CSF DC cultures responded with IFN production. Cells transfected with full length or subgenomic viral RNA stimulated IFN release indicating that infectious virus particle formation is not essential in this process. Use of differentiated DC from mice with genetic lesions in innate immune signalling showed that IFN secretion by HCV-stimulated murine DC was independent of MyD88 and CARDIF, but dependent on TRIF and IFNAR signalling. Separating Flt3-L DC cultures into pDC and conventional CD11b-like and CD8α-like DC revealed that the CD8α-like DC, homologous to the human CD141+ DC, release interferon upon stimulation by HCV replicating cells. In contrast, the other cell types and in particular the pDC did not. Injection of human HCV subgenomic replicon cells into IFN-β reporter mice confirmed the interferon induction upon HCV replication in vivo. These results indicate that HCV-replicating cells stimulate IFN secretion from murine CD8α-like DC independent of infectious virus production. Thus, this work defines basic principles of viral recognition by murine DC populations. Moreover, this model should be useful to explore the interaction between dendritic cells during HCV replication and to define how viral signatures are delivered to and recognized by immune cells to trigger IFN release.
Author Summary
HCV is an RNA virus that, following exposure, in most cases establishes chronic infection. The virus has evolved numerous immune evasion strategies, including direct interference with interferon production. Nevertheless, HCV infection activates interferon-stimulated genes in the liver, implying that non-infected cells secrete IFN. Several DC subsets have been implicated in HCV sensing and production of IFN; however, the molecular mechanism resulting in HCV sensing is poorly understood. Using murine bone marrow derived DC, we dissected basic principles of HCV innate immune recognition and activation of dendritic cells. We show that HCV recognition by murine DCs depends on TRIF and IFN receptor signalling. This indicated the involvement of TLR3 and of the IFN receptor dependent amplification loop. Infectious virus production is dispensable since cells carrying subgenomic HCV replicons are also recognized. Moreover, specific DC subtypes, i.e. CD8α-like DC, are responsible for recognition of HCV. These findings highlight that specific murine DC subpopulations are uniquely capable of recognizing HCV replicating cells independent of infectious virus production. These observations open novel opportunities to explore the mechanisms of inter-cellular communication that mediate activation and IFN production of non-infected immune cells and to dissect the role of DC subsets in immune control.
doi:10.1371/journal.ppat.1005736
PMCID: PMC4934921  PMID: 27385030
22.  Unsupervised High-Dimensional Analysis Aligns Dendritic Cells across Tissues and Species 
Immunity  2016;45(3):669-684.
Summary
Dendritic cells (DCs) are professional antigen-presenting cells that hold great therapeutic potential. Multiple DC subsets have been described, and it remains challenging to align them across tissues and species to analyze their function in the absence of macrophage contamination. Here, we provide and validate a universal toolbox for the automated identification of DCs through unsupervised analysis of conventional flow cytometry and mass cytometry data obtained from multiple mouse, macaque, and human tissues. The use of a minimal set of lineage-imprinted markers was sufficient to subdivide DCs into conventional type 1 (cDC1s), conventional type 2 (cDC2s), and plasmacytoid DCs (pDCs) across tissues and species. This way, a large number of additional markers can still be used to further characterize the heterogeneity of DCs across tissues and during inflammation. This framework represents the way forward to a universal, high-throughput, and standardized analysis of DC populations from mutant mice and human patients.
Graphical Abstract
Image 1
Highlights
•A conserved gating strategy aligns dendritic cells (DCs) in mouse and human tissues•Unsupervised computational analysis of flow cytometry data outperforms manual analysis•Mass cytometry reveals heterogeneity of DC subsets across mouse and human tissues•DC activation upon inflammation tracked by automated analysis of mass cytometry
Using unsupervised analysis of flow cytometry and mass cytometry data obtained from multiple mouse, macaque, and human tissues, Guilliams et al. provide a universal toolbox for the automated identification of dendritic cells. This framework represents the way forward to high-throughput and standardized analysis of dendritic cells from mutant mice and patients.
doi:10.1016/j.immuni.2016.08.015
PMCID: PMC5040826  PMID: 27637149
23.  The Signal Transducer STAT5 Inhibits Plasmacytoid Dendritic Cell Development by Suppressing Transcription Factor IRF8 
Immunity  2008;28(4):509-520.
SUMMARY
The development of distinct dendritic cell (DC) subsets is regulated by cytokines. Flt3-ligand- (Flt3L) is necessary for plasmacytoid (pDC) and conventional DC (cDC) maturation. GM-CSF inhibits Flt3L-driven pDC production while promoting cDC growth. We show that GM-CSF selectively utilizes STAT5 to block Flt3L-dependent pDC development from the lineage-negative, Flt3+ (lin−/Flt3+) bone marrow subset. STAT3, by contrast, is necessary for expansion of DC progenitors but not pDC maturation. In vivo, STAT5 suppresses pDC formation during repopulation of the DC compartment following bone marrow ablation. GM-CSF/STAT5 signaling rapidly extinguishes pDC-related gene expression in lin−/Flt3+ progenitors. Inspection of the Irf8 promoter revealed that STAT5 is recruited during GM-CSF-mediated suppression, indicating STAT5 directly inhibits transcription of this critical pDC gene. Our results therefore show that GM-CSF controls the production of pDCs by employing STAT5 to suppress IRF8 and the pDC transcriptional network in lin−/Flt3+ progenitors.
doi:10.1016/j.immuni.2008.02.013
PMCID: PMC2864148  PMID: 18342552
plasmacytoid dendritic cells; GM-CSF; STAT5; development; FLT3
24.  Dysfunctional DC subsets in RCC patients: Ex vivo correction to yield an effective anti-cancer vaccine 
Molecular immunology  2008;46(5):893-901.
Dendritic cells (DCs) are potent antigen-presenting cells responsible for the activation and functional polarization of specific T cells. In patients with renal cell carcinoma (RCC) and other cancers, coordinate DC and T cell defects have been reported. In particular, DC and T cell functional subsets that are not conducive to tumor clearance are hypothesized to predominate in patients with advanced-stage disease. Two major peripheral blood DC subsets have been identified in humans: myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) that are believed to mediate contrasting effects on cancer immunity.
Given the lack of information regarding DC subsets in patients with RCC, in the present study we have investigated the comparative frequencies and activation states of mDC and pDC in peripheral blood, cancer tissues and lymph nodes of patients with RCC using flow cytometry and immunohistochemistry. Three monoclonal antibodies (mAbs) reactive against specific DC subsets (BDCA-2 or BDCA-4 for pDC and BDCA-1 and BDCA-3 which represent two distinct subsets of mDC, mDC1 and mDC2, respectively) were employed. We observed a significant reduction of both DC subsets in the peripheral blood of patients as compared to normal donors. Similarly, both mDC and pDC were recruited in large numbers into RCC tumor tissues, where they displayed an immature phenotype (DC-LAMP−) and appeared unable to differentiate into mature DC (CD83+) that were competent to migrate to draining lymph nodes.
However, we were readily able to generate ex vivo mDC from RCC patients. These DC stimulated robust anti-tumor CTL in vitro and would be envisioned for use in DC-based vaccines applied in patients with RCC whose existing immune system is judged dysfunctional, anergic or prone to undergo apoptosis.
doi:10.1016/j.molimm.2008.09.015
PMCID: PMC3427923  PMID: 19041139
Renal cell carcinoma; dendritic cells; lymph nodes; confocal microscopy; T cell response
25.  The equivalents of human blood and spleen dendritic cell subtypes can be generated in vitro from human CD34+ stem cells in the presence of fms-like tyrosine kinase 3 ligand and thrombopoietin 
Dendritic cells (DCs) are immune cells specialized to capture, process and present antigen to T cells in order to initiate an appropriate adaptive immune response. The study of mouse DC has revealed a heterogeneous population of cells that differ in their development, surface phenotype and function. The study of human blood and spleen has shown the presence of two subsets of conventional DC including the CD1b/c+ and CD141+CLEC9A+ conventional DC (cDC) and a plasmacytoid DC (pDC) that is CD304+CD123+. Studies on these subpopulations have revealed phenotypic and functional differences that are similar to those described in the mouse. In this study, the three DC subsets have been generated in vitro from human CD34+ precursors in the presence of fms-like tyrosine kinase 3 ligand (Flt3L) and thrombopoietin (TPO). The DC subsets so generated, including the CD1b/c+ and CLEC9A+ cDCs and CD123+ pDCs, were largely similar to their blood and spleen counterparts with respect to surface phenotype, toll-like receptor and transcription factor expression, capacity to stimulate T cells, cytokine secretion and cross-presentation of antigens. This system may be utilized to study aspects of DC development and function not possible in vivo.
doi:10.1038/cmi.2012.48
PMCID: PMC4002222  PMID: 23085949
dendritic cells; fms-like tyrosine kinase 3 ligand; thrombopoietin

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