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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.
PMCID: PMC2882835  PMID: 20479118
2.  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.
PMCID: PMC1971124  PMID: 17850179
3.  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.
PMCID: PMC2880586  PMID: 20532161
4.  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.
PMCID: PMC3754054  PMID: 23785206
5.  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.
PMCID: PMC3992850  PMID: 24114554
Bacille Calmette-Guérin (BCG); BDCA-1+ myeloid DCs; CD8+ T cells; human; plasmacytoid DCs; tuberculosis
6.  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.
PMCID: PMC4113936  PMID: 24980046
7.  Continuous expression of the transcription factor E2-2 maintains the cell fate of mature plasmacytoid dendritic cells 
Immunity  2010;33(6):905-916.
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.
PMCID: PMC3010277  PMID: 21145760
8.  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.
PMCID: PMC3608935  PMID: 23497451
Plasmacytoid dendritic cells; Gene expression; Granzyme B
9.  The Signal Transducer STAT5 Inhibits Plasmacytoid Dendritic Cell Development by Suppressing Transcription Factor IRF8 
Immunity  2008;28(4):509-520.
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.
PMCID: PMC2864148  PMID: 18342552
plasmacytoid dendritic cells; GM-CSF; STAT5; development; FLT3
10.  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.
PMCID: PMC4002222  PMID: 23085949
dendritic cells; fms-like tyrosine kinase 3 ligand; thrombopoietin
11.  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.
PMCID: PMC3427923  PMID: 19041139
Renal cell carcinoma; dendritic cells; lymph nodes; confocal microscopy; T cell response
12.  Expression of the T Cell Receptor αβ on a CD123+ BDCA2+ HLA-DR+ Subpopulation in Head and Neck Squamous Cell Carcinoma 
PLoS ONE  2011;6(1):e15997.
Human Plasmacytoid Dendritic Cells (PDCs) infiltrating solid tumor tissues and draining lymph nodes of Head and Neck Squamous Cell Carcinoma (HNSCC) show an impaired immune response. In addition to an attenuated secretion of IFN-α little is known about other HNSCC-induced functional alterations in PDCs. Particular objectives in this project were to gain new insights regarding tumor-induced phenotypical and functional alterations in the PDC population. We showed by FACS analysis and RT-PCR that HNSCC orchestrates an as yet unknown subpopulation exhibiting functional autonomy in-vitro and in-vivo besides bearing phenotypical resemblance to PDCs and T cells. A subset, positive for the PDC markers CD123, BDCA-2, HLA-DR and the T cell receptor αβ (TCR-αβ) was significantly induced subsequent to stimulation with HNSCC in-vitro (p = 0.009) and also present in metastatic lymph nodes in-vivo. This subgroup could be functionally distinguished due to an enhanced production of IL-2 (p = 0.02), IL-6 (p = 0.0007) and TGF-β (not significant). Furthermore, after exposure to HNSCC cells, mRNA levels revealed a D-J-beta rearrangement of the TCR-beta chain besides a strong enhancement of the CD3ε chain in the PDC population. Our data indicate an interface between the PDC and T cell lineage. These findings will improve our understanding of phenotypical and functional intricacies concerning the very heterogeneous PDC population in-vivo.
PMCID: PMC3019173  PMID: 21264308
13.  Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells1 
Plasmacytoid dendritic cells (pDC) are rare cells found in peripheral blood and lymphoid tissues. pDC are considered to be “professional” type I interferon (IFN) producing cells and produce 10–100-fold more IFN-α than other cell types in response to enveloped viruses or synthetic TLR-7 and -9 agonists. In this study, purified pDC were found to express high levels of IFN-λ receptor mRNA as well as cell-surface IFN-λ receptor. We have developed intracellular flow cytometry assays using antibodies to IFN-λ1/3 or -λ2 to assess the expression of IFN-λ proteins by pDC. We observed that a subset of human pDC expresses only intracellular IFN-α while another subset produces both IFN-α and IFN-λ after stimulation with virus or the TLR9 agonist, CpGA; the cells that co-expressed IFN-α and IFN-λ were the cells with the highest levels of IFN-α expression. Antibody cross-linking of CD4 or BDCA-2 molecules on pDC inhibited both HSV-induced IFN-λ and IFN-α production. Like the production of IFN-α, the HSV-induced IFN-λ production in pDC was mediated through TLR9 and independent of virus replication. Exogenous IFN-λ treatment of pDC resulted in increased virus-induced expression of both IFN-α and IFN-λ. In addition, both exogenous IFN-λ and –α inhibited dexamethasone-induced apoptosis of pDC. We conclude that pDC are major producers of IFN-λ1 and –λ2 in response to viral stimulation and also express functional receptors for this cytokine. Thus, IFN-λ can serve as an autocrine signal to strengthen the antiviral response of pDC by increasing IFN-α and IFN-λ production, resulting in prolonged pDC survival.
PMCID: PMC3579503  PMID: 22891284
Human; dendritic cells; cytokines; cytokine receptors; viral infection
14.  miR-22 Controls Irf8 mRNA Abundance and Murine Dendritic Cell Development 
PLoS ONE  2012;7(12):e52341.
MicroRNAs (miRNAs) have emerged as critical regulators of many cellular responses, through the action of miRNA-induced silencing complex (miRISC)- or miRNA ribonucleoprotein complex (miRNP)-mediated gene repression. Here we studied the role of miRNAs in the development of dendritic cells (DCs), an important immune cell type that is divided into conventional DC (cDC) and plasmacytoid DC (pDC) subsets. We found that miR-22 was highly expressed in mouse CD11c+ CD11b+ B220− cDCs compared to pDCs, and was induced in DC progenitor cell cultures with GM-CSF, which stimulate CD11c+ CD11b+ B220− cDC differentiation. Enforced overexpression of miR-22 during DC development enhanced CD11c+ CD11b+ B220− cDC generation at the expense of pDCs, while miR-22 knockdown demonstrated opposite effects. Moreover, overexpression and knockdown of miR-22 showed significant effects on the mRNA abundance of Irf8, which encodes the transcription factor IRF8 that plays essential roles in DC development. Luciferase reporter assays confirmed that miR-22 binds directly to the 3′UTR of the mouse Irf8 mRNA. Collectively, these results suggest that miR-22 targets Irf8 mRNA for posttranscriptional repression and controls DC subset differentiation.
PMCID: PMC3522651  PMID: 23251709
15.  CD2AP/SHIP1 Complex Positively Regulates Plasmacytoid Dendritic Cell Receptor Signaling by Inhibiting the E3 Ubiquitin Ligase Cbl 
The human plasmacytoid dendritic cell (pDC) receptor BDCA2 forms a complex with the adaptor FcεR1γ to activate an ITAM-signaling cascade. BDCA2 receptor signaling negatively regulates the TLR7/9-mediated type 1 IFN responses in pDCs, which may play a key role in controlling self-DNA/RNA–induced autoimmunity. We report in this article that CD2-associated adaptor protein (CD2AP), which is highly expressed in human pDCs, positively regulates BDCA2/FcεR1γ receptor signaling. By immunoprecipitation and mass spectrometry analyses, we found that CD2AP bound to SHIP1. Knockdown of CD2AP or SHIP1 reduced the BDCA2/FcεR1γ-mediated ITAM signaling and blocked its inhibition of TLR9-mediated type 1 IFN production. Knockdown of CD2AP or SHIP1 also enhanced the ubiquitination and degradation of Syk and FcεR1γ that was mediated by the E3 ubiquitin ligase Cbl. This led us to discover that, upon BDCA2 cross-linking, the CD2AP/SHIP1 complex associated with Cbl and inhibited its E3 ubiquitin ligase activity. In human primary pDCs, cross-linking of the BDCA2/FcεR1γ complex induced the recruitment of the CD2AP/SHIP1/Cbl complex to the plasma membrane of pDCs, where it colocalized with the BDCA2/FcεR1γ complex. Therefore, CD2AP positively regulates BDCA2/FcεR1γ signaling by forming a complex with SHIP1 to inhibit the E3 ubiquitin ligase Cbl.
PMCID: PMC3665352  PMID: 22706086
16.  Characterization of conventional and atypical receptors for the chemokine CCL2 on mouse leukocytes 
Chemokine-directed leukocyte migration is crucial for effective immune and inflammatory responses. Conventional chemokine receptors (cCKRs) directly control cell movement, atypical chemokine receptors (ACKRs) regulate co-expressed cCKRs, and both cCKRs and ACKRs internalize chemokines to limit their abundance in vivo, a process referred to as scavenging. A leukocyte’s migratory and chemokine scavenging potential is determined by which cCKRs and ACKRs it expresses, and by the ligand specificity, signaling properties, and chemokine internalization capacity of these receptors. Most chemokines can bind at least one cCKR and one ACKR. CCL2 can bind to CCR2 (a cCKR) and two ACKRs (ACKR1 and ACKR2). Here, by using fluorescent CCL2 uptake to label cells bearing functional CCL2 receptors, we have defined the expression profile, scavenging activity, and ligand specificity of CCL2 receptors on mouse leukocytes. We show that qualitative and quantitative differences in the expression of CCR2 and ACKR2 endow individual leukocyte subsets with distinctive CCL2 receptor profiles and CCL2 scavenging capacities. We reveal that some cells, including plasmacytoid dendritic cells, can express both CCR2 and ACKR2; that Ly6Chi monocytes have particularly strong CCL2 scavenging potential in vitro and in vivo; and that CCR2 is a much more effective CCL2 scavenger than ACKR2. We confirm the unique, overlapping, ligand specificities of CCR2 and ACKR2, and, unexpectedly, find that cell context influences the interaction of CCL7 and CCL12 with CCR2. Fluorescent chemokine uptake assays were instrumental in providing these novel insights into CCL2 receptor biology, and the sensitivity, specificity and versatility of these assays is discussed.
PMCID: PMC4065784  PMID: 24890717
17.  Characterization of CD56+ Dendritic-Like Cells: A Normal Counterpart of Blastic Plasmacytoid Dendritic Cell Neoplasm? 
PLoS ONE  2013;8(11):e81722.
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematological malignancy. Plasmacytoid DCs (pDCs), which are defined as lineage marker (Lin)−HLA−DR+CD56−CD123+CD11c− cells, are considered to be the normal counterpart of BPDCNs. However, BPDCN can be distinguished from pDCs by uniform expression of CD56. In this study, to identify a normal counterpart of BPDCN, we searched for a Lin−HLA−DR+CD56+ population and focused on a minor subpopulation of Lin−DR+CD56+CD123+CD11c− cells that we designated as pDC-like cells (pDLCs). pDLC constituted 0.03% of peripheral blood mononuclear cells (PBMCs), and the pDLC/pDC ratio was higher in bone marrow cells than in PBMCs. pDLC clearly expressed BDCA2, BDCA4, and myeloid antigens, which are frequently expressed by BPDCN. pDLCs exhibited modest expression of Toll-like receptors and produced less interferon-α after CpG stimulation, but presented very low endocytic ability unlike mDCs. These functional differences were attributed to the expression profile of transcriptional factors. After in vitro culture with Flt3-ligand and GM-CSF, pDLCs expressed CD11c and BDCA1. These data suggested that pDLCs are a distinct subpopulation, with an immunophenotype similar to BPDCNs. Moreover, our results indicate that pDLCs might be immature DCs and might contribute to the immunophenotypical diversity of BPDCNs.
PMCID: PMC3843704  PMID: 24312342
18.  Similar antigen cross-presentation capacity and phagocytic functions in all freshly isolated human lymphoid organ–resident dendritic cells 
The Journal of Experimental Medicine  2013;210(5):1035-1047.
Tonsil-resident BDCA1+ DCs, BDCA3+ DCs, and pDCs all cross-present antigen efficiently.
Dendritic cells (DCs) represent a heterogeneous population of antigen-presenting cells that initiate and orient immune responses in secondary lymphoid organs. In mice, lymphoid organ–resident CD8+ DCs are specialized at cross-presentation and have developed specific adaptations of their endocytic pathway (high pH, low degradation, and high export to the cytosol). In humans, blood BDCA3+ DCs were recently shown to be the homologues of mouse CD8+ DCs. They were also proposed to cross-present antigens more efficiently than other blood DC subsets after in vitro activation, suggesting that in humans cross-presentation is restricted to certain DC subsets. The DCs that cross-present antigen physiologically, however, are the ones present in lymphoid organs. Here, we show that freshly isolated tonsil-resident BDCA1+ DCs, BDCA3+ DCs, and pDCs all cross-present soluble antigen efficiently, as compared to macrophages, in the absence of activation. In addition, BDCA1+ and BDCA3+ DCs display similar phagosomal pH and similar production of reactive oxygen species in their phagosomes. All three DC subsets, in contrast to macrophages, also efficiently export internalized proteins to the cytosol. We conclude that all freshly isolated lymphoid organ–resident human DCs, but not macrophages, display high intrinsic cross-presentation capacity.
PMCID: PMC3646495  PMID: 23569327
19.  Superior antigen cross-presentation and XCR1 expression define human CD11c+CD141+ cells as homologues of mouse CD8+ dendritic cells 
The Journal of Experimental Medicine  2010;207(6):1273-1281.
In recent years, human dendritic cells (DCs) could be subdivided into CD304+ plasmacytoid DCs (pDCs) and conventional DCs (cDCs), the latter encompassing the CD1c+, CD16+, and CD141+ DC subsets. To date, the low frequency of these DCs in human blood has essentially prevented functional studies defining their specific contribution to antigen presentation. We have established a protocol for an effective isolation of pDC and cDC subsets to high purity. Using this approach, we show that CD141+ DCs are the only cells in human blood that express the chemokine receptor XCR1 and respond to the specific ligand XCL1 by Ca2+ mobilization and potent chemotaxis. More importantly, we demonstrate that CD141+ DCs excel in cross-presentation of soluble or cell-associated antigen to CD8+ T cells when directly compared with CD1c+ DCs, CD16+ DCs, and pDCs from the same donors. Both in their functional XCR1 expression and their effective processing and presentation of exogenous antigen in the context of major histocompatibility complex class I, human CD141+ DCs correspond to mouse CD8+ DCs, a subset known for superior antigen cross-presentation in vivo. These data define CD141+ DCs as professional antigen cross-presenting DCs in the human.
PMCID: PMC2882837  PMID: 20479115
20.  Differential response of BDCA-1+ and BDCA-3+ myeloid dendritic cells to respiratory syncytial virus infection 
Respiratory Research  2013;14(1):71.
Respiratory syncytial virus (RSV) is the leading cause of respiratory infections in children, elderly, and immunocompromised individuals. Severe infection is associated with short- and long-term morbidity including pneumonia, recurrent wheezing, and abnormal pulmonary function, and several lines of evidence indicate that impaired adaptive immune responses during infection are critical in the pathophysiology of RSV-mediated disease. Myeloid Dendritic cells (mDCs) play a pivotal role in shaping antiviral immune responses in the respiratory tract; however, few studies have examined the interactions between RSV and individual mDC subsets. In this study, we examined the effect of RSV on the functional response of primary mDC subsets (BDCA-1+ and BDCA-3+) isolated from peripheral blood.
BDCA-1+ and BDCA-3+ mDCs were isolated from the peripheral blood of healthy adults using FACS sorting. Donor-matched BDCA-1+ and BDCA-3+ mDCs were infected with RSV at a multiplicity of infection (MOI) of 5 for 40 hours. After infection, cells were analyzed for the expression of costimulatory molecules (CD86, CD80, and PD-L1), cytokine production, and the ability to stimulate allogenic CD4+ T cell proliferation.
Both BDCA-1+ and BDCA-3+ mDCs were susceptible to infection with RSV and demonstrated enhanced expression of CD86, and the inhibitory costimulatory molecules CD80 and PD-L1. Compared to BDCA-3+ mDCs, RSV-infected BDCA-1+ mDC produced a profile of cytokines and chemokines predominantly associated with pro-inflammatory responses (IL-1β, IL-6, IL-12, MIP-1α, and TNF-α), and both BDCA-1+ and BDCA-3+ mDCs were found to produce IL-10. Compared to uninfected mDCs, RSV-infected BDCA-1+ and BDCA-3+ mDCs demonstrated a reduced capacity to stimulate T cell proliferation.
RSV infection induces a distinct pattern of costimulatory molecule expression and cytokine production by BDCA-1+ and BDCA-3+ mDCs, and impairs their ability to stimulate T cell proliferation.
The differential expression of CD86 and pro-inflammatory cytokines by highly purified mDC subsets in response to RSV provides further evidence that BDCA-1+ and BDCA-3+ mDCs have distinct roles in coordinating the host immune response during RSV infection. Findings of differential expression of PD-L1 and IL-10 by infected mDCs, suggests possible mechanisms by which RSV is able to impair adaptive immune responses.
PMCID: PMC3708742  PMID: 23829893
RSV; Myeloid dendritic cells; BDCA-1+ mDCs; BDCA-3+ mDCs; Primary DCs; Immune response; Costimulatory molecules; CD80; CD86; PD-L1; Cytokine profiles
21.  Antigen delivery to early endosomes eliminates the superiority of human blood BDCA3+ dendritic cells at cross presentation 
The Journal of Experimental Medicine  2013;210(5):1049-1063.
Human BDCA3 DCs are superior to BDCA1 DCs at antigen cross presentation when delivered to late endosomes and lysosomes but not when delivered to early endosomes.
Human BDCA3+ dendritic cells (DCs), the proposed equivalent to mouse CD8α+ DCs, are widely thought to cross present antigens on MHC class I (MHCI) molecules more efficiently than other DC populations. If true, it is unclear whether this reflects specialization for cross presentation or a generally enhanced ability to present antigens on MHCI. We compared presentation by BDCA3+ DCs with BDCA1+ DCs using a quantitative approach whereby antigens were targeted to distinct intracellular compartments by receptor-mediated internalization. As expected, BDCA3+ DCs were superior at cross presentation of antigens delivered to late endosomes and lysosomes by uptake of anti-DEC205 antibody conjugated to antigen. This difference may reflect a greater efficiency of antigen escape from BDCA3+ DC lysosomes. In contrast, if antigens were delivered to early endosomes through CD40 or CD11c, BDCA1+ DCs were as efficient at cross presentation as BDCA3+ DCs. Because BDCA3+ DCs and BDCA1+ DCs were also equivalent at presenting peptides and endogenously synthesized antigens, BDCA3+ DCs are not likely to possess mechanisms for cross presentation that are specific to this subset. Thus, multiple DC populations may be comparably effective at presenting exogenous antigens to CD8+ T cells as long as the antigen is delivered to early endocytic compartments.
PMCID: PMC3646496  PMID: 23569326
22.  Plasmacytoid Dendritic Cells Suppress HIV-1 Replication but Contribute to HIV-1 Induced Immunopathogenesis in Humanized Mice 
PLoS Pathogens  2014;10(7):e1004291.
The role of plasmacytoid dendritic cells (pDC) in human immunodeficiency virus type 1 (HIV-1) infection and pathogenesis remains unclear. HIV-1 infection in the humanized mouse model leads to persistent HIV-1 infection and immunopathogenesis, including type I interferons (IFN-I) induction, immune-activation and depletion of human leukocytes, including CD4 T cells. We developed a monoclonal antibody that specifically depletes human pDC in all lymphoid organs in humanized mice. When pDC were depleted prior to HIV-1 infection, the induction of IFN-I and interferon-stimulated genes (ISGs) were abolished during acute HIV-1 infection with either a highly pathogenic CCR5/CXCR4-dual tropic HIV-1 or a standard CCR5-tropic HIV-1 isolate. Consistent with the anti-viral role of IFN-I, HIV-1 replication was significantly up-regulated in pDC-depleted mice. Interestingly, the cell death induced by the highly pathogenic HIV-1 isolate was severely reduced in pDC-depleted mice. During chronic HIV-1 infection, depletion of pDC also severely reduced the induction of IFN-I and ISGs, associated with elevated HIV-1 replication. Surprisingly, HIV-1 induced depletion of human immune cells including T cells in lymphoid organs, but not the blood, was reduced in spite of the increased viral replication. The increased cell number in lymphoid organs was associated with a reduced level of HIV-induced cell death in human leukocytes including CD4 T cells. We conclude that pDC play opposing roles in suppressing HIV-1 replication and in promoting HIV-1 induced immunopathogenesis. These findings suggest that pDC-depletion and IFN-I blockade will provide novel strategies for treating those HIV-1 immune non-responsive patients with persistent immune activation despite effective anti-retrovirus treatment.
Author Summary
Persistent expression of IFN-I is correlated with disease progression in HIV-1 infected humans or SIV-infected monkeys. Thus, persistent pDC activation has been implicated in contributing to AIDS pathogenesis. To define the role of pDC in HIV-1 infection and immunopathogenesis in vivo, we developed a monoclonal antibody that specifically and efficiently depletes human pDC in all lymphoid organs in humanized mice. We discover that pDC are the critical IFN-I producer cells in response to acute HIV-1 infection, because depletion of pDC completely abolished induction of IFN-I or ISG by HIV-1 infection, correlated with elevated level of HIV-1 replication. When pDC were depleted during chronic HIV-1 infection in humanized mice, pDC were still the major IFN-I producing cells in vivo, which contributed to HIV-1 suppression. Despite of higher level of viral replication in pDC-depleted mice, we found that HIV-induced depletion of human T cells and leukocytes was significantly reduced in lymphoid organs, correlated with reduced cell death induction by HIV-1 infection. Our findings demonstrate that pDC play two opposing roles in HIV-1 pathogenesis: they produce IFN-I to suppress HIV-1 replication and induce death of human immune cells to contribute to HIV-induced T cell depletion and immunopathogenesis.
PMCID: PMC4117636  PMID: 25077616
23.  Human blood dendritic cells from allergic subjects have impaired capacity to produce interferon-α via TLR9 
High affinity IgE receptor (FcεRI) expression on blood dendritic cells reportedly correlates with serum IgE levels. Our studies demonstrate that plasmacytoid dendritic cells (pDCs) secrete pro-inflammatory cytokines (IL-6, TNF-α) following FcεRI stimulation – a mode of activation that simultaneously reduces expression of Toll-like receptor 9 (TLR9). Whether or not TLR9 and/or FcεRI levels and their function on dendritic cells relate to allergic status is unknown.
The aim of this study is to compare the innate (TLR9-mediated) immune response of human plasmacytoid dendritic cells to TLR9 and FcεRIα receptor expression in allergic and nonallergic subjects.
Basophil depleted mononuclear cell fractions containing pDCs were prepared from peripheral blood of allergic and non-allergic subjects. Intracellular TLR9 and surface FcεRIα expression in BDCA-2 positive cells were determined by flow cytometry. Activating anti-IgE antibody, anti-FcεRIα antibody, and TLR9 agonist were used to stimulate cell suspensions, with cytokine levels determined by ELISA.
No difference in the frequency of pDCs was detected among allergic (n = 9) versus nonallergic (n = 11) subjects (P = 0.261). While there was also no difference in baseline expression of TLR9, pDCs from allergic subjects produced 6-fold less IFN-α when stimulated with CpG (P = 0.002). Conversely, there was higher FcεRIα expression (P = 0.01) on the pDCs of allergic subjects.
Impaired TLR9-dependent immune responses in human plasmacytoid dendritic cells are associated with allergic status and inversely correlate with FcεRIα expression. This impaired innate immune response among dendritic cells of allergic subjects may lead to more targeted therapeutic approaches and could provide a better understanding of the mechanisms underlying conventional and CpG-based immunotherapy.
PMCID: PMC2707903  PMID: 18318750
allergy; dendritic cells; IFN-α; plasmacytoid; TLR9; CpG-DNA; cytokines
24.  Expression of High-Affinity IgE Receptor on Human Peripheral Blood Dendritic Cells in Children 
PLoS ONE  2012;7(2):e32556.
In a mouse model of viral induced atopic disease, expression of FcεRI on dendritic cells is critical. While adult human conventional (cDC) and plasmacytoid (pDC) dendritic cells have been shown to express FcεRI, it is not known if this receptor is expressed in childhood and how its expression is governed by IgE.
Following informed consent of subjects (n = 27, aged 12–188 months), peripheral blood was stained for surface expression of CD19, ILT7, CD1c, IgE, FcεRI and analyzed by flow cytometry (cDC: CD19− ILT7− CD1c+; pDC: CD19− ILT7+ CD1c−). Total and specific serum IgE levels to food and inhalant allergens were determined by ImmunoCAP, and the relationship between FcεRI expression on dendritic cells and sensitization, free IgE, cell bound IgE, and age was determined.
Independent of sensitization status, FcεRI expression was noted on cDC and pDC as early as 12 months of age. Serum IgE level correlated with expression of FcεRI on cDC, but not pDC. Based on the concentration of IgE, a complex relationship was found between surface bound IgE and expression of FcεRI on cDC. pDC exhibited a linear relationship of FcεRI expression and bound IgE that was consistent through all IgE concentrations.
In children, FcεRI expression on cDC and pDC is modulated differently by serum and cell bound IgE. IgE governance of FcεRI expression on cDC depends upon a complex relationship. Further studies are needed to determine the functional roles of FcεRI on cDC and pDC.
PMCID: PMC3285694  PMID: 22384272
25.  Immune Adjuvant Efficacy of CpG Oligonucleotide in Cancer Treatment Is Founded Specifically upon TLR9 Function in Plasmacytoid Dendritic Cells 
Cancer research  2011;71(20):6428-6437.
The differences in function, location, and migratory pattern of conventional dendritic cells (cDC) and plasmacytoid DCs (pDC) not only point to specialized roles in immune responses but also signify additive and interdependent relationships required to clear pathogens. We studied the in vivo requirement of cross-talk between cDCs and pDCs for eliciting antitumor immunity against in situ released tumor antigens in the absence or presence of the Toll-like receptor (TLR) 9 agonist CpG. Previous data indicated that CpG boosted tumor-specific T-cell responses after in vivo tumor destruction and increased survival after tumor rechallenges. The present study shows that cDCs are indispensable for cross-presentation of ablation-released tumor antigens and for the induction of long-term antitumor immunity. Depletion of pDCs or applying this model in type I IFN receptor–deficient mice abrogated CpG-mediated responses. CD8α+ cDCs and the recently identified merocytic cDCs were dependent on pDCs for CpG-induced upregulation of CD80. Moreover, DC transfer studies revealed that merocytic cDCs and CD8α+ cDCs were most susceptible to pDC help and subsequently promoted tumor-free survival in a therapeutic setting. By transferring wild-type pDCs into TLR9-deficient mice, we finally showed that TLR9 expression in pDCs is sufficient to benefit from CpG as an adjuvant. These studies indicate that the efficacy of CpG in cancer immunotherapy is dependent on cross-talk between pDCs and specific subsets of cDCs.
PMCID: PMC3653311  PMID: 21788345

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