Advertisement
  • Loading metrics

BDCA2/FcεRIγ Complex Signals through a Novel BCR-Like Pathway in Human Plasmacytoid Dendritic Cells

  • Wei Cao ,

    To whom correspondence should be addressed. E-mail: wcao@mdacc.tmc.edu (WC); yjliu@mdacc.tmc.edu (YJL).

    Affiliation Department of Immunology, University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America

  • Li Zhang,

    Affiliation Department of Biostatistics and Applied Mathematics, University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America

  • David B Rosen,

    Affiliations Department of Microbiology and Immunology, University of California at San Francisco, San Francisco, California, United States of America , Biomedical Sciences Graduate Program, University of California at San Francisco, San Francisco, California, United States of America , Cancer Research Institute, University of California at San Francisco, San Francisco, California, United States of America

  • Laura Bover,

    Affiliation Department of Immunology, University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America

  • Gokuran Watanabe,

    Affiliation Department of Immunology, University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America

  • Musheng Bao,

    Affiliation Department of Immunology, University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America

  • Lewis L Lanier,

    Affiliations Department of Microbiology and Immunology, University of California at San Francisco, San Francisco, California, United States of America , Biomedical Sciences Graduate Program, University of California at San Francisco, San Francisco, California, United States of America , Cancer Research Institute, University of California at San Francisco, San Francisco, California, United States of America

  • Yong-Jun Liu

    To whom correspondence should be addressed. E-mail: wcao@mdacc.tmc.edu (WC); yjliu@mdacc.tmc.edu (YJL).

    Affiliation Department of Immunology, University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America

BDCA2/FcεRIγ Complex Signals through a Novel BCR-Like Pathway in Human Plasmacytoid Dendritic Cells

  • Wei Cao, 
  • Li Zhang, 
  • David B Rosen, 
  • Laura Bover, 
  • Gokuran Watanabe, 
  • Musheng Bao, 
  • Lewis L Lanier, 
  • Yong-Jun Liu
PLOS
x

Abstract

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 FcεRIγ. 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 FcεRIγ, 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 FcεRIγ. 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 FcεRIγ, 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.

Introduction

Dendritic cells (DCs) are specialized sentinels in the immune system that detect invading pathogens and pathological damages of the host. Upon activation, DCs instruct appropriate and effective immune responses [13]. Their extraordinary ability to capture antigens (self or foreign) is largely mediated by the collective expression of C-type lectin receptors (CLRs) on the cell surface [4,5]. In mouse spleen, DC subsets display a discrete expression profile of C-type lectins—CD8+ DCs express DEC-205 (CD205), whereas CD8 DCs express dendritic cell immunoreceptor 2 (DCIR2), dendritic cell immunoactivating receptor (DCAR), Dectin-1, Dectin-2, DCIR3, and DC-SIGN [6]. This expression profile, in conjunction with the polarized expression of antigen processing and presentation machinery, contributes to the intrinsic functional difference between the DC subsets in regulating immunity [6]. In humans, C-type lectin ICAM3-grabbing nonintegrin (DC-SIGN, CD209) is expressed by mucosal DCs and langerin (CD207) is expressed by Langerhans cells [3,4]. Besides tissue DCs, macrophage, myeloid DCs (mDCs) and monocytes express a number of CLRs, such as Dectin-1, macrophage mannose receptor (MMR), and DCIR [3,4].

Plasmacytoid DCs (pDCs), also referred to as natural type I interferon (IFN)-producing cells (IPCs), are a distinct DC population with an extraordinary ability to rapidly produce massive amounts of type I IFN in response to viral infections [7,8]. Blood dendritic cell antigen 2 (BDCA2), a C-type lectin uniquely expressed on resting human pDCs, has been used to unequivocally identify pDCs in human peripheral blood and tissues [9]. It has an intracellular N terminus and only one extracellular carbohydrate recognition domain, which belongs to the type II C-type lectin group [5,10,11]. Similar to CLRs on other DC subsets, BDCA2 has been suggested to be capable of functioning as an antigen-uptake receptor, because monoclonal antibodies (mAbs) bound to BDCA2 on the surface of pDCs are efficiently internalized, and mAb-derived peptide bound to MHC class II can be presented to T cells [10].

Resting pDCs uniquely express high amounts of Toll-like receptor 9 (TLR9) and TLR7 [1214], which recognize microbial DNA or RNA ligands in the endo-lysosomal compartment, respectively. After activation of TLR7 or TLR9, a multi-component “cytoplasmic transductional–transcriptional complex” is formed to initiate cellular activations [1517]. Type I IFN production by pDCs has been shown to be independent of RNA helicases retinoic acid–inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and their adaptor IFN-ß promoter stimulator-1 (IPS-1) [15,1820], which are essential for IFN response to viruses by other immune or nonhematopoietic cells. Therefore, pDCs carry out their unique innate immune functions through the TLR-mediated pathway. After exposure to viruses, pDCs differentiate into efficient antigen-presenting DCs and thus act as a critical mediator linking innate and adaptive immune responses against viral infections [7,8]. However, aberrant pDC activation in the absence of infection has been associated with autoimmune diseases. Patients with active systemic lupus erythematosus (SLE) have high amounts of type I IFN in their sera, which is likely due to the activation of pDCs by autoantibodies complexed with self nuclear antigens [2124]. Because both TLR7 and TLR9 are located intracellularly, how pDCs sense the extracellular microenvironment and, in turn, modulate these TLR-mediated responses remains an important question.

BDCA2 was the first receptor described that negatively regulates the IFN response of pDCs, which is induced by CpG oligonucleotide, influenza virus, or by autoantibodies complexed with DNA [25,26]. However, it is not clear how BDCA2 modulates the TLR signaling, because its cytoplasmic domain does not contain any known signaling motifs, even though BDCA2 ligation by mAbs causes protein tyrosine phosphorylation and calcium influx [25]. A recent analysis of the closely related dendritic cell immunoreceptor family lectins, which include DCIR, DCAR, Dectin-2, and BDCA2, suggests a potential shared signaling mechanism as a result of the similar arrangement of their transmembrane domains [11,27,28]. Both DCAR and Dectin-2 have been shown to associate with FcεRIγ to transduce cellular activation signals [29,30].

Here we report that BDCA2 signals in pDCs by associating with the transmembrane adapter FcεRIγ and initiates an immunoreceptor-based tyrosine activation motif (ITAM)–dependent signaling cascade. We also provide evidence that human pDCs express a series of signaling molecules known to be specifically involved in BCR activation. We demonstrate that the BDCA2 and FcεRIγ complex activates a prominent BCR-like signaling pathway in human pDCs to modulate the TLR-mediated responses.

Results

BDCA2 Associates with and Signals through FcεRIγ

To determine the expression profile of BDCA2 in human leukocytes, we searched our established gene expression database, which includes the major immune cell types in peripheral blood. Strikingly, BDCA2 transcripts were expressed abundantly and exclusively by human pDCs (Figure 1A), consistent with its specific surface expression on pDCs within total peripheral blood mononuclear cell (PBMC) [9]. By contrast, immature peripheral blood CD11c+ mDC, monocyte, and monocyte-derived DC (mono-DC) expressed transcripts of other C-type lectins, such as DC-SIGN, Dectin-1, DCIR, and MMR, which were absent in pDCs (Figure 1A).

thumbnail
Figure 1. BDCA2 Associates with FcεRIγ To Form a pDC-Specific Receptor Complex

(A) The expression profile of BDCA2 transcripts in peripheral blood leukocytes by microarray gene expression analysis is shown. In comparison, the relative expression levels of DC-SIGN, Dectin-1, DCIR, and MMR are also shown. A value of <1 (arbitrary units) indicates the absence of gene expression.

(B) Surface expression of BDCA2 after transduction of BDCA2 into Jurkat cells previously transfected with control, FcεRIγ, DAP12, DAP10, or a Y-F ITAM mutant FcεRIγ. The surface staining by isotype-matched control mAb is shown as a shaded area.

(C) Co-immunoprecipitation of BDCA2 and FcεRIγ from BDCA2 and FcεRIγ doubly transfected Jurkat cells after cell surface biotinylation. Western blot analysis was performed with anti-FcεRIγ Ab or with HRP-conjugated NeutrAvidin.

(D) Co-immunoprecipitation of BDCA2 and FcεRIγ from freshly isolated pDCs. Western blot analysis was performed with anti-FcεRIγ Ab.

https://doi.org/10.1371/journal.pbio.0050248.g001

pDCs express two transmembrane adapter proteins bearing ITAMs, i.e., FcεRIγ and DAP12, as well as DAP10, which signals via a YINM motif to activate PI3K [31]. To test whether BDCA2 interacts with any of these adapters, the individual adapters were first stably transduced into Jurkat cells, a human T cell line. When BDCA2 was transfected into Jurkat cells containing FcεRIγ, the expression of BDCA2 on the cell surface was greatly enhanced, indicating a potential interaction between BDCA2 and FcεRIγ (Figure 1B). In contrast, the presence of DAP12 and DAP10 only marginally affected BDCA2 expression, whereas both DAP12 and DAP10 increased the surface expression of mouse NKG2D, used as a positive control [31]. The enhanced BDCA2 surface expression in the presence of FcεRIγ was also observed in several T and B cell lines (Figure S1). To demonstrate directly the physical association between BDCA2 and FcεRIγ, we performed co-immunoprecipitation experiments and showed that FcεRIγ existed in a complex that could be precipitated by antibody to BDCA2, but not by an isotype-matched control antibody from transfected Jurkat cells (Figure 1C) and, furthermore, from freshly isolated human pDCs (Figure 1D). These data suggest that BDCA2 and FcεRIγ form a receptor complex with signaling potential.

Previously, BDCA2-transfected Jurkat cells were shown to be unable to initiate protein tyrosine phosphorylation after BDCA2 crosslinking, an observation suggesting the potential involvement of a signaling adapter [25]. When BDCA2 was crosslinked on the Jurkat cells that were cotransfected with different adapters, Jurkat cells expressing FcεRIγ, but not DAP12 or DAP10, prominently phosphorylated a spectrum of proteins (Figure 2A). We constructed a mutant FcεRIγ in which the tyrosine (Y) residue within the intracellular ITAM was replaced with phenylalanine (F), rendering it unable to recruit downstream molecules. This mutant FcεRIγ supported the elevated levels of surface expression of BDCA2 (Figure 1B) but was unable to mediate the protein tyrosine phosphorylation after BDCA2 crosslinking (Figure 2B), indicating an essential role of the ITAM in FcεRIγ for the signaling by the BDCA2/FcεRIγ complex.

thumbnail
Figure 2. Crosslinking the BDCA2/FcεRIγ Complex Transduces Activation Signals

(A) Total protein phosphorylation in Jurkat cells transfected with BDCA2 in the absence or presence of adapter proteins by anti-BDCA2 mAb or control IgG1 crosslinking. Western blot analysis was performed with anti-phosphotyrosine mAb PY20.

(B) Total protein phosphorylation induced by anti-BDCA2 mAb crosslinking in Jurkat cells transfected with BDCA2 and wild-type (WT) or mutant (Y-to-F) FcεRIγ.

(C) Transfected mouse 2B4 T hybridoma cells with an NFAT-GFP reporter (transgenes are labeled on the top) were crosslinked with control mouse IgG1 or anti-BDCA2 mAb. Cells were analyzed for GFP expression by flow cytometry. Cells cultured in medium alone or with ionomycin (positive control) were also analyzed.

(D) The kinetics of intracellular calcium flux in transfected Jurkat cells (transgenes are labeled on top) when crosslinked (marked by arrow) by control (IgG1) or anti-BDCA2 mAb.

https://doi.org/10.1371/journal.pbio.0050248.g002

Furthermore, when a human BDCA2/FcεRIγ complex was introduced into a mouse T cell hybridoma line that contained an intracellular nuclear factor of activated T cells–green fluorescent protein (NFAT-GFP) reporter construct [31,32], crosslinking BDCA2 by mAb resulted in GFP expression (Figure 2C), indicating that the BDCA2/FcεRIγ receptor complex was able to activate NFAT, similar to the effect of TCR-induced activation. In the absence of FcεRIγ, crosslinking BDCA2 did not induce GFP expression (Figure 2C). Finally, calcium influx, one of the important cellular activation events that occurs downstream of ITAM signaling, was effectively triggered by BDCA2 crosslinking in Jurkat cells transfected with both BDCA2 and FcεRIγ, but not in cells transfected with BDCA2 alone (Figure 2D), further confirming the requirement of FcεRIγ for BDCA2 to signal.

Human pDCs Express Signaling Molecules Involved in BCR Activation

To further delineate the intracellular signaling events following BDCA2 and FcεRIγ activation, we examined the expression by pDCs of components known to be involved in ITAM-mediated signaling. In lymphocytes, several well-established signaling pathways exist downstream of the B cell and T cell antigen receptors, linked through ITAM-bearing subunits of these receptor complexes. We analyzed the expression of molecules involved in BCR and TCR activation in pDCs in comparison with peripheral B cells, T cells, natural killer (NK) cells, mDCs, and monocytes.

After receptor activation, two core tyrosine residues within the ITAM(s) are phosphorylated by Src family protein tyrosine kinases (PTKs). The phosphorylated tyrosines within the ITAMs associate with the src homology 2(SH2) domains of Syk-family PTKs, which in turn phosphorylate cell-type–specific intracellular adapters to initiate a multitude of signaling events. Each type of lymphocyte expresses and uses a distinct set of proteins to carry out the receptor-proximal signal transduction [33,34]. B cells express B lymphoid tyrosine kinase (BLK), Lyn kinase, spleen tyrosine kinase (Syk), and two adapter molecules, BLNK and BCAP, whereas T cells preferentially use two other Src-family PTKs, Fyn and Lck, and CD3ξ-associated protein kinase (ZAP-70) from the Syk-family of PTKs, as well as alternative adapters, i.e., SLP-76 and linker for activation of T cells (LAT) (Figure 3A). Strikingly, pDCs express many members of the BCR signaling cascade, but not those involved in TCR activation (Figure 3A). Of note, the levels of the B cell–specific adapter B cell linker (BLNK, also known as SLP-65 or BASH) was transcribed at higher levels in pDC than in B cells (514 relative units versus 133 relative units, respectively). Another B cell adapter, BCAP, which mediates phosphoinositide-3-kinase (PI3K) signaling [35], was also abundantly expressed by pDCs (Figure 3A). pDCs expressed lower levels of BLK compared to B cells. Interestingly, NK cells, in addition to expressing several members of the TCR signaling pathway, were found also to express several molecules involved in BCR signaling, e.g., Lyn, Syk and BCAP. mDC and monocyte similarly express Lyn, Syk, BCAP, and Fyn as well as the T cell adaptor SLP76. However unlike pDCs and B cells, they both lack the expression of BLNK and BLK (Figure 3A). Within all the peripheral cell leukocytes we examined, BLNK transcripts were only transcribed by pDCs and B cells (unpublished data).

thumbnail
Figure 3. Human pDCs Express a BCR-Like Signaling Cascade

(A) Expression of genes encoding molecules involved in BCR (top panel) and TCR (bottom panel) signaling in six peripheral leukocyte cell types from microarray gene expression analysis.

(B) The relative gene expression of signaling molecules in different cell types from three healthy donors determined by quantitative RT-PCR analysis. The expression was normalized with the level of the transcript in total PBMC. The median expression is marked by a horizontal bar.

(C) The expression of signaling proteins in different cell types was determined by Western blot analysis. The results from one representative donor out of three healthy donors analyzed are shown.

https://doi.org/10.1371/journal.pbio.0050248.g003

We next analyzed the expression of the signaling components operating downstream of the molecules described above. pDCs and B cells selectively transcribed PLCγ2, Vav2, and Vav3, which are absent in T cells (Figure S2). On the other hand, high levels of PKCθ are abundantly expressed by T and NK cells, but not by pDCs or B cells (Figure S5). In contrast, the expression of other downstream signaling molecules, such as PI3K, Nck, MAP kinases, and NFAT, failed to show a distinct cell type–specific pattern (Figure S2).

We confirmed the transcription and protein expression of the BCR proximal signaling molecules in pDCs from several healthy donors by RT-PCR and Western blot analysis (Figure 3B and 3C). Taken together, our analysis revealed the existence of a potential ITAM-mediated signaling cascade in human pDCs similar to that of B cells, but distinct from that in T cells, NK cells, monocytes, or mDCs (Figure 4).

thumbnail
Figure 4. Diagram of ITAM-Mediated Signaling Pathways in Human Leukocytes

The schematic cascades that are involved in receptor proximal signaling (solid arrows) and those leading to NF-κB activation (dotted lines) are shown.

https://doi.org/10.1371/journal.pbio.0050248.g004

BDCA2/FcεRIγ Complex Transduces Signals via a BCR-Like Signaling Cascade

To investigate whether the BDCA2/FcεRIγ receptor complex could signal through the BCR signaling cascade, Namalwa cells, a human Burkitt lymphoma cell line, was transfected with BDCA2 and FcεRIγ. Similar to BCR activation, crosslinking of the BDCA2/FcεRIγ complex on Namalwa cells resulted in rapid phosphorylation of residue Y525 of Syk and residue Y416 of Src-family PTKs (Figure 5). After BCR activation, five tyrosine residues on BLNK are phosphorylated, enabling its function as a scaffold to integrate and propagate signals to downstream proteins [36]. BDCA2 ligation resulted in phosphorylation of residue Y84 of BLNK (Figure 4). Additional downstream molecules, such as Vav1 and PLCγ2, were phosphorylated by BDCA2 crosslinking, similar to BCR activation (Figure 5). MAP kinases Erk1/2, which are also activated after BCR engagement through the Ras-Raf pathway, were rapidly phosphorylated after BDCA2 stimulation (Figure 5). Furthermore, we observed a robust Ca++ influx in Namalwa cells after BDCA2 crosslinking, which is dependent on FcεRIγ (unpublished data).

thumbnail
Figure 5. BDCA2/FcεRIγ Complex Signals through the BCR Signaling Cascade in Transfected B Cells

Shown are the kinetics of tyrosine phosphorylation of BCR intracellular signaling components in transduced Namalwa cells when crosslinked by control IgG1 or anti-BDCA2 mAb in comparison with BCR activation by anti-human Ig Ab.

https://doi.org/10.1371/journal.pbio.0050248.g005

To study the signaling of the BDCA2/FcεRIγ complex in primary human pDCs, we crosslinked BDCA2 by mAbs on freshly isolated pDCs and confirmed that it potently induces a transient intracellular calcium flux (Figure 6A). This activity was inhibited by PP2, a compound that inhibits Src family PTKs, but was not affected by an inactive control compound, PP3. In addition, Syk inhibitor completely abolished the intracellular calcium flux in BDCA2-crosslinked pDCs (Figure 6A). These results suggest the functional involvement of both Syk and Src-family PTKs during BDCA2 signaling. When the phosphorylation status of key signaling molecules in pDCs was analyzed, BDCA2 crosslinking resulted in rapid phosphorylation of both Src family PTKs and Syk (Figure 6B), indicating the onset of ITAM-induced signaling in pDCs. Different from the results with the Namalwa cell line, BDCA2 crosslinking did not enhance the pre-existing phosphorylation of BLNK in pDCs (Figure 6B, lane 5). High levels of phosphorylation of Y84 in the BLNK protein have been observed in freshly isolated, resting pDCs from several healthy donors, but not in freshly isolated human B cells (unpublished data). Moreover, downstream molecules, including Vav1, PLCγ2, and Erk1/2, were phosphorylated by BDCA2 crosslinking on pDCs (Figure 6B). In contrast, crosslinking with neither the isotype-matched control antibody nor the mAb against BDCA-4, another surface molecule expressed on pDCs, phosphorylated these proteins under the same conditions (Figure 6B). It should be noted that BDCA-4 beads were used to isolate pDCs, therefore BDCA-4 may no longer available for efficient crosslinking. The Src PTK inhibitor PP2 or Syk inhibitor, but not PP3, greatly diminished BDCA2-induced phosphorylation of both Src family PTKs and Syk and blocked the phosphorylation of Vav1 and PLCγ2 (Figure S3). Thus, the BDCA2/FcεRIγ complex signals through a BCR-like signaling cascade in primary human pDCs.

thumbnail
Figure 6. BDCA2/FcεRIγ Complex Signals through a BCR-Like Signaling Cascade in Primary pDCs

(A) The kinetics of intracellular calcium flux in freshly isolated pDCs when crosslinked (marked by arrow) by control IgG1 or anti-BDCA2 Ab. As indicated, cells were pre-incubated with PP2, PP3, or Syk inhibitor prior to crosslinking.

(B) The kinetics of tyrosine phosphorylation on BCR intracellular signaling components in freshly isolated pDCs when crosslinked by control IgG1, anti-BDCA2 mAb, or anti-BDCA-4 mAb.

https://doi.org/10.1371/journal.pbio.0050248.g006

BDCA2/FcεRIγ Complex Signaling Inhibits TLR-Mediated Type I IFN Production

Although BCR signaling synergizes with TLR triggering for optimal B lymphocyte activation [37], the impact of this pathway on TLR-mediated type I IFN responses needs to be defined. To accomplish this, we stably transfected human Burkitt lymphoma Namalwa cells with IRF7, BDCA2 and FcεRIγ. These cells with endogenous TLR9 expression [38] produced IFNα in response to stimulation with CpG oligonucleotide (ODN) (Figure 7A). Crosslinking of BDCA2/FcεRIγ or BCR together with CpG activation in these Namalwa cells resulted in enhanced interleukin 10 (IL-10) production, as expected, but decreased IFNα secretion (Figure 7A), indicating a selective suppressive effect by BCR or BDCA2 activation on TLR-induced IFNα production.

thumbnail
Figure 7. Ligation of the BDCA2/FcεRIγ Complex Inhibits TLR-Induced Type I IFN Production

(A) Transfected Namalwa cells were stimulated with CpG ODN and ligated with anti-BDCA2, anti-BCR, or control mAbs. Shown are the amounts of IFNα and IL-10 secreted by the cells stimulated CpG in a triplicate assay.

(B) Purified pDCs were stimulated by CpG (top panel) or R848 (bottom panel) following anti-BDCA2 or control mAb crosslinking. The amounts of the secreted IFNα, TNFα, and IL-6 in a triplicate assay from a representative donor (n ≥ 3) are shown.

(C) Purified pDCs stimulated by CpG first and then treated with anti-BDCA2 or control mAbs at different time intervals. The amounts of the secreted IFNα, TNFα, and IL-6 from a representative donor (n ≥ 3) are shown.

(D) The amounts of type I IFN transcripts were determined by quantitative RT-PCR analysis. The expression is shown as the relative level of transcription compared with unstimulated PBMC.

https://doi.org/10.1371/journal.pbio.0050248.g007

To investigate the function of the BCR-like signaling cascade in pDCs, we analyzed, in detail, the impact of BDCA2 crosslinking on the innate immune responses of human primary pDCs. Purified pDCs were stimulated with CpG ODN 2216 or R848, ligands of TLR9 and TLR7 or TLR8, respectively, in the presence of anti-BDCA2 mAb or control mAbs. BDCA2 ligation potently inhibited IFNα production and invariably reduced the production of tumor necrosis factor α (TNFα) and IL-6 induced by stimulation with these TLR ligands (Figure 7B). pDCs secreted lower levels of IFNα after BDCA2 ligation, but normal amounts of chemokines CXCL10/IP-10, CCL3/MIP-1α and CCL4/MIP-1β in response to CpG (Figure S4).

To evaluate the kinetics of BDCA2-mediated suppression of IFN production, pDCs were first stimulated with CpG and control or anti-BDCA2 mAbs were added to the culture at different times afterward. Strikingly, up to 4 h after pDCs were exposed to CpG ODN, crosslinking BDCA2 by mAb effectively inhibited IFNα and cytokine production by pDCs (Figure 7C). This result demonstrates the potency of BDCA2 to inhibit the TLR-induced innate immune responses by pDCs. Independently, pDCs from multiple donors were stimulated simultaneously with both CpG and anti-BDCA2 or control mAbs and at different lapsed times, the amounts of IFNα in the supernatant were measured (Figure S5). At 4 h after CpG treatment, pDCs treated with anti-BDCA2 mAbs reduced the production of IFNα by 96%. By 8 h post CpG treatment, BDCA2-ligated pDCs produced an average of 25.6% of IFNα compared to the amount made by control IgG1-incubated pDCs (Figure S5). Therefore, BDCA2 crosslinking likely blocked the initial induction of IFNα secretion.

To further determine the stage of IFN suppression, we measured the amount of type I IFN transcripts from CpG-activated pDCs that were crosslinked with anti-BDCA2 or control mAbs. BDCA2 crosslinking uniformly reduced the transcription of all the subtypes of type I IFN analyzed, i.e., IFNα, IFNβ, IFNγ, and IFNω (Figure 7D). Therefore, BDCA2 ligation blocks the transcriptional of type I IFN by pDCs in response to TLR activation.

Discussion

To date, BDCA2 is the only C-type lectin that is found expressed exclusively on human pDCs, and it serves as a defining marker for pDCs. In addition, BDCA2 represents one of the most potent receptors that modulates the ability of pDCs to elicit type I IFN responses. Here we provide experimental evidence to reveal the signaling mechanism used by BDCA2 in human pDCs. On the cell surface, BDCA2 associates with FcεRIγ to form a signaling receptor complex, which, upon ligation, activates a novel BCR-like intracellular signaling cascade, interfering with TLR activation.

ITAM-based signaling is the primary pathway of activation used by classical immunoreceptors, such as the antigen receptors on B and T lymphocytes, NK cell receptors, and the Fc receptors on myeloid cells [3941]. ITAM-mediated signaling is also used by additional receptors, such as cell adhesion molecules, chemokine receptors, plexins, and lectin receptors [42]. pDC and mDCs represent two major subsets of professional antigen-presenting cells in human peripheral blood and tissues, which use different sets of pattern recognition receptors, such as TLRs, CLRs, and Nod-like receptors, for sensing and responding to microbial infections [4350]. Our present study suggests that mDCs and pDCs not only express different repertories of lectin receptors, but also suggests that the signal pathways used by CLRs bearing ITAM elements on mDC and pDCs are intrinsically different. Ligation of either the BDCA2/FcεRIγ complex or certain myeloid cell lectins, such as Dectin-1 and DC-SIGN [51,52], phosphorylates Syk, which is involved in BCR membrane-proximal signaling. However, downstream of Syk, pDCs use the intracellular adaptor molecule BLNK, whereas mDCs use SLP76 for amplification of signaling (Figure 7). In addition, recent studies showed that CARD9, a signaling adaptor molecule, plays a critical role in the signal transduction of Dectin-1 and ITAM-associated receptors in myeloid cells [53,54]. Our analysis suggests that similar to lymphocytes, pDCs do not express CARD9, but likely use CARMA1 (also named CARD11) to activate nuclear factor κB (NF-κB) [5557] (Figure S6 and Figure 7).

Previous studies have shown remarkable similarities between B lineage cells and pDCs, including their plasma cell morphology and shared expression of several classes of genes. For example, they express a similar sets of TLRs (TLR7 and TLR9) [12,14,58], possess germ-line immunoglobulin (Ig) transcripts and Ig recombinase [5961], have B cell–related transcription factor Spi-B [59,62], and use the same CIITA promoter for the controlling of MHC Class II expression [63]. The present demonstration that the pDC receptor BDCA2/FcεRIγ and BCR share the same membrane-proximal signaling cascade and pathway leading to NF-κB activation provides further support for the intriguing relationship between pDCs and B lymphocytes.

A central mediator during B cell activation, BLNK, is phosphorylated by BCR activation on five tyrosine residues (Y72, Y84, Y96, Y178, and Y189) that serve as docking sites for recruiting downstream molecules [34,36]. Interestingly, in resting primary pDCs, we observed high levels of, if not saturating, phosphorylation of Y84 in BLNK, which is involved in PLCγ2 activation [36]. Further examination of tyrosine phosphorylation on other residues of BLNK may shed light on the dynamic involvement of this key molecule in nucleating signaling components during BDCA2 activation.

A recent study reported that BDCA2 may serve as an alternative receptor, in addition to CD4, CXCR4, and CCR5 on human pDCs, for HIV via binding to gp120 [64]. Therefore, it is plausible that HIV uptake by BDCA2 negatively affects the ability of pDCs to mount type I IFN responses. Similarly, Mycobacterium tuberculosis targets DC-specific C-type lectin DC-SIGN to infect DCs and to directly down-regulate DC-mediated immune responses [65]. Moreover, measles virus (MV) interacts with CD46, its cellular receptor on human monocytes, to specifically down-regulate IL-12 production, a mechanism causing MV-induced immunosuppression [66]. Therefore, BDCA2 may represent another example of antigen uptake receptors targeted by microbial ligands to negatively regulate cellular immunity.

Paradoxically, activation by antigens through the BCR triggers B cell proliferation, differentiation, and Ig and cytokine production, whereas crosslinking of BDCA2, which activates a similar BCR-like signaling pathway, fails to stimulate pDCs to proliferate or produce cytokines [10] (W. Cao, unpublished data), but rather inhibits the TLR-mediated responses. As multiple receptors on pDCs apparently use ITAM-based mechanisms to modulate type I IFN and cytokine responses during TLR activation [17,31,6770], our findings will facilitate further investigations to determine how TLR activation is regulated in pDCs. Such mechanisms may provide opportunities for therapeutic interventions in autoimmune diseases, such as SLE [24], Sjögren syndrome [71,72], polymyositis [73,74], and psoriasis [75,76], where the causative mechanisms might involve hyper-activation of pDCs.

Materials and Methods

Media, cell lines, and reagents.

RPMI-1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 ng/ml streptomycin (Invitrogen; http://www.invitrogen.com), and heat-inactivated 10% FBS (Atlanta Biologicals; http://www.atlantabio.com) were used for the cell culture. The human acute T cell leukemia cell line Jurkat and the Burkitt lymphoma cell line Namalwa were purchased from the American Type Culture Collection (ATCC). The mouse 2B4 T hybridoma cell line was generously provided by H. Arase, Osaka University, Japan. CpG ODN 2216 (GGGGGACGATCGTCGG-GGGG and CpG 2006 (TCGTCGTTTTGTCGT-TTTGTCGTT) (Qiagen; http://www.qiagen.com) and R848 (InVivogen; http://www.invivogen.com) were used for pDC stimulation.

Immune cell isolation and establishment of gene expression database.

The institutional review board for human research at the M.D. Anderson Cancer Center approved this study. Our gene expression database containing multiple types of immune cells isolated from human peripheral blood was established as described [31]. For this study, four peripheral leukocyte cell types were isolated simultaneously from buffy coats of individual healthy donors. Briefly, pDCs were positively selected from PBMC with anti-BDCA-4-coated microbeads and sorted by flow cytometry as CD3CD4+CD8CD11cCD14CD16CD19CD56. The remaining PBMCs were stained and individual cell types were sorted accordingly: B cells (CD4CD8CD11cCD14CD16CD19+CD56CD235aBDCA2), CD4+ T cells (CD4+CD8CD11cCD14CD16CD19CD56CD235aBDCA2), and NK cells (CD3CD4CD8CD14CD19CD56+CD235aBDCA2).

Quantitative real-time PCR.

Total RNA extraction and quantitative real-time (RT)-PCR were performed as described [31]. Oligonucleotide primers used are listed in the Table S1 or described previously [31].

Cloning and expression of BDCA2 and transmembrane adapters.

Full-length human BDCA2 was cloned from cDNAs generated from human peripheral blood pDCs by high-fidelity PCR using the following primers: sense, ATATGGATCCATGGTGCCTGAAGAA-GAGC and antisense, CTATGAATTCTTATATGT-AGATCTTCTTCATC. The BDCA2 cDNA was then cloned into the Bam HI and Eco RI sites of a lentiviral vector FG-30 [77]. Full-length cDNA encoding human FcεRIγ, DAP12, and DAP10, as described previously [31], were subcloned into FG-30. An FcεRIγ mutant with Y-to-F substitution in the ITAM was prepared by PCR and cloned into the FG-30 lentiviral vector for gene transduction.

NFAT-GFP reporter cell assays.

Both BDCA2 and FcεRIγ, or each cDNA individually, were transduced into mouse 2B4 T hybridoma cells stably expressing an NFAT-GFP reporter construct [32]. Transfected cells were cultured at 106 cells/ml in the presence of 10 μg/ml of plate-bound anti-BDCA2 mAb AC144 (Miltenyi Biotec; http://www.miltenyibiotec.com) or purified IgG1 isotype-matched control mAb (eBioscience; http://www.ebioscience.com) for 20 h and then analyzed for GFP expression by flow cytometry.

Cell surface biotinylation and protein immunoprecipitation.

2 × 107 cells that were pre-washed with phosphate-buffered saline (PBS) were incubated with 2 mM EZ-Link Sulfo-NHS-LC-Biotin (Pierce Biotechnology; http://www.piercenet.com) according to manufacturer's protocol. Cells were then washed 3 time with 100 mM glycine in 1× PBS, and lysed in Brij-NP-40 lysis buffer (0.875% Brij 97, 0.125% Nonidet P-40, 10 mM Tris base, pH 8.0, 150 mM NaCl plus protease inhibitors). Then the lysates were cleared by a spin at 10,000g for 10 min. The lysates were incubated with either control mouse IgG1 or anti-BDCA2 mAb conjugated to Protein G-Sepharose (Pierce Biotechnology) at 4 °C overnight. The sepharose beads were washed extensively with the lysis buffer before elution of the antigen with SDS sample buffer. Western blots were performed with rabbit anti- FcεRIγ antibody (gift from Dr. M-H Jouvin, BIDMC, Boston) and HRP-conjugated goat anti-rabbit IgG or HRP-labeled neutravidin (Pierce Biotechnology).

Western blot analysis of protein tyrosine phosphorylation and expression.

5 × 105 cells transfected with BDCA2 and FcεRIγ were incubated with control mouse IgG1 (eBioscience) or anti-BDCA2 mAb (Miltenyi Biotec), and then crosslinked with F(ab')2 goat anti-mouse IgG (Jackson ImmunoResearch Lab; http://www.jacksonimmuno.com). BCR crosslinking was performed by adding 15 μg/ml F(ab')2 goat anti-human Ig (Southern Biotech). 5 × 105 pDCs isolated using a BDCA-4 cell isolation kit (Miltenyi Biotec) were incubated with control mouse IgG1, anti-BDCA2, or anti-BDCA-4 mAb (Miltenyi Biotec). Western blot was performed with anti-phospho Src family Ab, anti-phospho Syk Ab, anti-phospho Erk1/2, anti-non-phospho Src family Ab, anti-Syk Ab (Cell Signaling Technology; http://www.cellsignal.com), anti-phospho BLNK, anti-phospho Vav1 (Abcam; http://www.abcam.com), anti-phospho PLCγ2 (BD Biosciences; http://www.bdbiosciences.com), and anti-β-actin mAb (Sigma; http://www.sigmaaldrich.com).

Calcium influx assay.

106 transfected cells or pDCs, pre-loaded with Fluo4- and Fura Red-AM (Invitrogen), were crosslinked as described above and analyzed on a FACSAria (BD Biosciences) and data were evaluated by using FlowJo software (TreeStar; http://www.treestar.com). As indicated, cells were pre-incubated for 30 min with 25 μM PP2, 25 μM PP3, or 5 μM Syk inhibitor (EMD Biosciences; http://www.emdbiosciences.com) prior to crosslinking.

Namalwa transfectants.

Namalwa cells were transduced sequentially by lentiviral vectors expressing human IRF7, BDCA2, and FcεRIγ. Full-length human IRF7 was cloned from cDNAs generated from purified human peripheral pDCs using a high-fidelity Taq polymerase (Invitrogen) with primers; sense, ACCTCTAGAATGGCCTTGGCTCCTGAGAGG, and antisense, ATTCTCGAGCTAGGCGGGCTGCTCCAGCTCC. The transduced Namalwa cells were stimulated with CpG ODN 2006 and then cultured in the presence of 10 μg/ml of plate-bound anti-BDCA2 mAb AC144, F(ab')2 goat anti-human Ig, or purified control IgG1 for 20 h. Supernatants were analyzed for cytokine secretion by ELISA using a human IFNα kit (Bender MedSystems; http://www.bendermedsystems.com) and IL-10 kit (R&D Systems; http://www.rndsystems.com).

BDCA2 crosslinking on pDCs in culture.

5 × 104 pDCs isolated by enrichment with anti-BDCA-4–coated microbeads and subsequent flow cytometry (CD4+CD11cCD3CD14CD16CD19CD56) were incubated with 5 μg/ml anti-BDCA2 or control mAb in 100 μl culture medium for 30 min prior to stimulation with 1 μM CpG 2216 or 0.05 μg/ml of R848. Cells and supernatants were harvested 18 h later for real-time quantitative PCR analysis and enzyme-linked immunosorbent assay (ELISA) using a human IFNα kit (Bender MedSystems), IFNα kit (PBL Biomedical Laboratories; http://www.interferonsource.com), IL-6 kit, TNFα kit, CXCL10 kit, CCL3 kit, and CCL4 kit (R&D Systems). For kinetic studies, pDCs were stimulated with 1 μM CpG 2216 and co-cultured with anti-BDCA2 or control mAbs at time 0. At different time points, supernatants were harvested for quantitation of cytokines or IFN by ELISA. Alternatively, CpG was added first to pDC cultures, and then anti-BDCA2 or control mAbs were added at different times thereafter. Eighteen h after addition of CpG, supernatants were harvested for quantitation of cytokines or IFN by ELISA.

Supporting Information

Figure S1. Expression Levels of BDCA2 in the Presence and Absence of FcεRIγ on Transfected T Cells (Mouse 2B4 T Hybridoma and Human Jurkat Cell Lines) and Transfected B Cells (Human HPB-Null Cell Line)

https://doi.org/10.1371/journal.pbio.0050248.sg001

(79 KB PPT)

Figure S2. Relative Transcription of Intracellular Molecules Involved in Antigen Receptor Signaling in Four Lymphoid Cell Types Based on Microarray Gene Expression Analysis

https://doi.org/10.1371/journal.pbio.0050248.sg002

(57 KB PPT)

Figure S3. Blocking of Tyrosine Phosphorylation of BCR-Like Intracellular Signaling Components Induced by BDCA2 Crosslinking by Inhibitors of Src PTK or Syk in Freshly Isolated pDCs

https://doi.org/10.1371/journal.pbio.0050248.sg003

(276 KB PDF)

Figure S4. Crosslinking of BDCA2 Inhibits TLR-Induced IFNβ but Not Chemokine Production

Purified pDCs were stimulated by CpG following anti-BDCA2 or control Ab crosslinking. The amounts of secreted IFNβ, CXCL10/IP-10, CCL3/MIP-1α and CCL4/MIP-1β from a representative donor (n ≥ 3) are shown.

https://doi.org/10.1371/journal.pbio.0050248.sg004

(49 KB PPT)

Figure S5. Purified pDCs Were Co-Stimulated by CpG and mAbs (Anti-BDCA2 or Control IgG) for 4 or 8 Hours

The relative amounts of the secreted IFNα from four donors are shown. The median expression is marked by a horizontal bar. The numbers on top of the symbols indicate the average amounts of IFNα (pg/ml) present in the culture supernatant.

https://doi.org/10.1371/journal.pbio.0050248.sg005

(44 KB PPT)

Figure S6. Relative Transcription of Intracellular Molecules Involved in NF-κB Activation in Peripheral Immune Cells Based on Microarray Gene Expression Analysis

https://doi.org/10.1371/journal.pbio.0050248.sg006

(49 KB PPT)

Table S1. Oligonucleotide Primers Used

https://doi.org/10.1371/journal.pbio.0050248.st001

(26 KB DOC)

Acknowledgments

The authors are grateful to Karen Ramirez, Zhiwei He, and Eric Wieder for cell sorting and technical support. We also thank Dr. Xiaofeng Qin for help on lentivirus constructions. LLL is an American Cancer Society Research Professor. DBR is supported by a Genentech Graduate Student Fellowship and a UCSF Chancellor's Research Fellowship.

Author Contributions

WC conceived, designed, and performed the experiments. LZ analyzed the data. DRB and GW performed some experiments. LB and MB prepared reagents. WC, LLL, and YJL had extensive scientific discussion for this study and wrote the paper.

References

  1. 1. Mellman I, Steinman RM (2001) Dendritic cells: Specialized and regulated antigen processing machines. Cell 106: 255–258.I. MellmanRM Steinman2001Dendritic cells: Specialized and regulated antigen processing machines.Cell106255258
  2. 2. Banchereau J, Steinman RM (1998) Dendritic cells and the control of immunity. Nature 392: 245–252.J. BanchereauRM Steinman1998Dendritic cells and the control of immunity.Nature392245252
  3. 3. Steinman R, Hemmi H (2006) Dendritic cells: Translating innate to adaptive immunity. Curr Top Microbiol Immunol 311: 17–58.R. SteinmanH. Hemmi2006Dendritic cells: Translating innate to adaptive immunity.Curr Top Microbiol Immunol3111758
  4. 4. Geijtenbeek TBH, van Vliet SJ, Engering A, t Hart BA, van Kooyk Y (2004) Self- and nonself-recognition by C-type lectins on dendritic cells. Annu Rev Immunol 22: 33–54.TBH GeijtenbeekSJ van VlietA. EngeringBA t HartY. van Kooyk2004Self- and nonself-recognition by C-type lectins on dendritic cells.Annu Rev Immunol223354
  5. 5. Cambi A, Figdor CG (2003) Dual function of C-type lectin-like receptors in the immune system. Curr Opin Cell Biol 15: 539–546.A. CambiCG Figdor2003Dual function of C-type lectin-like receptors in the immune system.Curr Opin Cell Biol15539546
  6. 6. Dudziak D, Kamphorst AO, Heidkamp GF, Buchholz VR, Trumpfheller C, et al. (2007) Differential antigen processing by dendritic cell subsets in vivo. Science 315: 107–111.D. DudziakAO KamphorstGF HeidkampVR BuchholzC. Trumpfheller2007Differential antigen processing by dendritic cell subsets in vivo.Science315107111
  7. 7. Colonna M, Trinchieri G, Liu YJ (2004) Plasmacytoid dendritic cells in immunity. Nature Immunol 5: 1219–1226.M. ColonnaG. TrinchieriYJ Liu2004Plasmacytoid dendritic cells in immunity.Nature Immunol512191226
  8. 8. Liu Y-J (2005) IPC: Professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors. Annu Rev Immunol 23: 275–306.Y-J Liu2005IPC: Professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors.Annu Rev Immunol23275306
  9. 9. Dzionek A, Fuchs A, Schmidt P, Cremer S, Zysk M, et al. (2000) BDCA-2, BDCA-3, and BDCA-4: Three markers for distinct subsets of dendritic cells in human peripheral blood. J Immunol 165: 6037–6046.A. DzionekA. FuchsP. SchmidtS. CremerM. Zysk2000BDCA-2, BDCA-3, and BDCA-4: Three markers for distinct subsets of dendritic cells in human peripheral blood.J Immunol16560376046
  10. 10. Dzionek A (2001) BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates antigen capture and is a potent inhibitor of interferon a/b induction. J Exp Med 194: 1823–1834.A. Dzionek2001BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates antigen capture and is a potent inhibitor of interferon a/b induction.J Exp Med19418231834
  11. 11. Kanazawa N (2007) Dendritic cell immunoreceptors: C-type lectin receptors for pattern-recognition and signaling on antigen-presenting cells. J Derm Sci 45: 77–86.N. Kanazawa2007Dendritic cell immunoreceptors: C-type lectin receptors for pattern-recognition and signaling on antigen-presenting cells.J Derm Sci457786
  12. 12. Kadowaki N, Ho S, Antonenko S, de Waal Malefyt R, Kastelein RA, et al. (2001) Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens. J Exp Med 194: 863–870.N. KadowakiS. HoS. AntonenkoR. de Waal MalefytRA Kastelein2001Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens.J Exp Med194863870
  13. 13. Jarrossay D, Napolitani G, Colonna M, Sallusto F, Lanzavecchia A (2001) Specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells. Eur J Immunol 31: 3388–3393.D. JarrossayG. NapolitaniM. ColonnaF. SallustoA. Lanzavecchia2001Specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells.Eur J Immunol3133883393
  14. 14. Hornung V, Rothenfusser S, Britsch S, Krug A, Jahrsdorfer B, et al. (2002) Quantitative expression of toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol 168: 4531–4537.V. HornungS. RothenfusserS. BritschA. KrugB. Jahrsdorfer2002Quantitative expression of toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides.J Immunol16845314537
  15. 15. Kawai T, Akira S (2006) Innate immune recognition of viral infection. Nat Immunol 7: 131–137.T. KawaiS. Akira2006Innate immune recognition of viral infection.Nat Immunol7131137
  16. 16. Honda K, Yanai H, Takaoka A, Taniguchi T (2005) Regulation of the type I IFN induction: a current view. Int Immunol 17: 1367–1378.K. HondaH. YanaiA. TakaokaT. Taniguchi2005Regulation of the type I IFN induction: a current view.Int Immunol1713671378
  17. 17. Cao W, Liu Y-J (2007) Innate immune functions of plasmacytoid dendritic cells. Curr Opin Immunol 19: 24–30.W. CaoY-J Liu2007Innate immune functions of plasmacytoid dendritic cells.Curr Opin Immunol192430
  18. 18. Stetson DB, Medzhitov R (2006) Antiviral defense: Interferons and beyond. J Exp Med. DB StetsonR. Medzhitov2006Antiviral defense: Interferons and beyond.J Exp Medjem.20061377. jem.20061377.
  19. 19. Kato H, Sato S, Yoneyama M, Yamamoto M, Uematsu S, et al. (2005) Cell type-specific involvement of RIG-I in antiviral response. Immunity 23: 19–28.H. KatoS. SatoM. YoneyamaM. YamamotoS. Uematsu2005Cell type-specific involvement of RIG-I in antiviral response.Immunity231928
  20. 20. Sun Q, Sun L, Liu H-H, Chen X, Seth RB, et al. (2006) The specific and essential role of MAVS in antiviral innate immune responses. Immunity 24: 633–642.Q. SunL. SunH-H LiuX. ChenRB Seth2006The specific and essential role of MAVS in antiviral innate immune responses.Immunity24633642
  21. 21. Baechler EC, Gregersen PK, Behrens TW (2004) The emerging role of interferon in human systemic lupus erythematosus. Curr Opin Immunol 16: 801–807.EC BaechlerPK GregersenTW Behrens2004The emerging role of interferon in human systemic lupus erythematosus.Curr Opin Immunol16801807
  22. 22. Vollmer J, Tluk S, Schmitz C, Hamm S, Jurk M, et al. (2005) Immune stimulation mediated by autoantigen binding sites within small nuclear RNAs involves Toll-like receptors 7 and 8. J Exp Med 202: 1575–1585.J. VollmerS. TlukC. SchmitzS. HammM. Jurk2005Immune stimulation mediated by autoantigen binding sites within small nuclear RNAs involves Toll-like receptors 7 and 8.J Exp Med20215751585
  23. 23. Means T, Luster A (2005) Toll-like receptor activation in the pathogenesis of systemic lupus erythematosus. Ann N Y Acad Sci 1062: 242–251.T. MeansA. Luster2005Toll-like receptor activation in the pathogenesis of systemic lupus erythematosus.Ann N Y Acad Sci1062242251
  24. 24. Pascual V, Banchereau J, Palucka A (2003) The central role of dendritic cells and interferon-alpha in SLE. Curr Opin Rheumatol 15: 548–556.V. PascualJ. BanchereauA. Palucka2003The central role of dendritic cells and interferon-alpha in SLE.Curr Opin Rheumatol15548556
  25. 25. Dzionek A, Sohma Y, Nagafune J, Cella M, Colonna M, et al. (2001) BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates antigen capture and is a potent inhibitor of interferon a/b induction. J Exp Med 194: 1823–1834.A. DzionekY. SohmaJ. NagafuneM. CellaM. Colonna2001BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates antigen capture and is a potent inhibitor of interferon a/b induction.J Exp Med19418231834
  26. 26. Dzionek A, Inagaki Y, Okawa K, Nagafune J, Rèock J, et al. (2002) Plasmacytoid dendritic cells: From specific surface markers to specific cellular functions. Hum Immunol 63: 1133–1148.A. DzionekY. InagakiK. OkawaJ. NagafuneJ. Rèock2002Plasmacytoid dendritic cells: From specific surface markers to specific cellular functions.Hum Immunol6311331148
  27. 27. Kanazawa N, Tashiro K, Miyachi Y (2004) Signaling and immune regulatory role of the dendritic cell immunoreceptor (DCIR) family lectins: DCIR, DCAR, dectin-2 and BDCA-2. Immunobiology 209: 179–190.N. KanazawaK. TashiroY. Miyachi2004Signaling and immune regulatory role of the dendritic cell immunoreceptor (DCIR) family lectins: DCIR, DCAR, dectin-2 and BDCA-2.Immunobiology209179190
  28. 28. Kanazawa N, Tashiro K, Inaba K, Lutz MB, Miyachi Y (2004) Molecular cloning of human Dectin-2. J Invest Dermatol 122: 1522–1524.N. KanazawaK. TashiroK. InabaMB LutzY. Miyachi2004Molecular cloning of human Dectin-2.J Invest Dermatol12215221524
  29. 29. Kanazawa N, Tashiro K, Inaba K, Miyachi Y (2003) Dendritic cell immunoactivating receptor, a novel C-type lectin immunoreceptor, acts as an activating receptor through association with Fc receptor g chain. J Biol Chem 278: 32645–32652.N. KanazawaK. TashiroK. InabaY. Miyachi2003Dendritic cell immunoactivating receptor, a novel C-type lectin immunoreceptor, acts as an activating receptor through association with Fc receptor g chain.J Biol Chem2783264532652
  30. 30. Sato K, Yang X-l, Yudate T, Chung J-S, Wu J, et al. (2006) Dectin-2 is a pattern recognition receptor for fungi that couples with the Fc receptor γ chain to induce innate immune responses. J Biol Chem 281: 38854–38866.K. SatoX-l YangT. YudateJ-S ChungJ. Wu2006Dectin-2 is a pattern recognition receptor for fungi that couples with the Fc receptor γ chain to induce innate immune responses.J Biol Chem2813885438866
  31. 31. Cao W, Rosen DB, Ito T, Bover L, Bao M, et al. (2006) Plasmacytoid dendritic cell-specific receptor ILT7-FceRIg inhibits Toll-like receptor-induced interferon production. J Exp Med 203: 1399–1405.W. CaoDB RosenT. ItoL. BoverM. Bao2006Plasmacytoid dendritic cell-specific receptor ILT7-FceRIg inhibits Toll-like receptor-induced interferon production.J Exp Med20313991405
  32. 32. Arase H, Mocarski ES, Campbell AE, Hill AB, Lanier LL (2002) Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors. Science 296: 1323–1326.H. AraseES MocarskiAE CampbellAB HillLL Lanier2002Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors.Science29613231326
  33. 33. Jun JE, Goodnow CC (2003) Scaffolding of antigen receptors for immunogenic versus tolerogenic signaling. Nat Immunol 4: 1057–1064.JE JunCC Goodnow2003Scaffolding of antigen receptors for immunogenic versus tolerogenic signaling.Nat Immunol410571064
  34. 34. Koretzky GA, Abtahian F, Silverman MA (2006) SLP76 and SLP65: Complex regulation of signalling in lymphocytes and beyond. Nat Rev Immunol 6: 67–78.GA KoretzkyF. AbtahianMA Silverman2006SLP76 and SLP65: Complex regulation of signalling in lymphocytes and beyond.Nat Rev Immunol66778
  35. 35. Okada T, Maeda A, Iwamatsu A, Gotoh K, Kurosaki T (2000) BCAP: The tyrosine kinase substrate that connects B cell receptor to phosphoinositide 3-kinase activation. Immunity 13: 817–827.T. OkadaA. MaedaA. IwamatsuK. GotohT. Kurosaki2000BCAP: The tyrosine kinase substrate that connects B cell receptor to phosphoinositide 3-kinase activation.Immunity13817827
  36. 36. Chiu C, Dalton M, Ishiai M, Kurosaki T, Chan A (2002) BLNK: Molecular scaffolding through 'cis'-mediated organization of signaling proteins. EMBO J 21: 6461–6472.C. ChiuM. DaltonM. IshiaiT. KurosakiA. Chan2002BLNK: Molecular scaffolding through 'cis'-mediated organization of signaling proteins.EMBO J2164616472
  37. 37. Krieg AM, Yi A-K, Matson S, Waldschmidt TJ, Bishop GA, et al. (1995) CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374: 546–549.AM KriegA-K YiS. MatsonTJ WaldschmidtGA Bishop1995CpG motifs in bacterial DNA trigger direct B-cell activation.Nature374546549
  38. 38. Henault M, Lee LN, Evans GF, Zuckerman SH (2005) The human Burkitt lymphoma cell line Namalwa represents a homogenous cell system characterized by high levels of Toll-like receptor 9 and activation by CpG oligonucleotides. J Immunol Methods 300: 93–99.M. HenaultLN LeeGF EvansSH Zuckerman2005The human Burkitt lymphoma cell line Namalwa represents a homogenous cell system characterized by high levels of Toll-like receptor 9 and activation by CpG oligonucleotides.J Immunol Methods3009399
  39. 39. Humphrey M, Lanier L, Nakamura M (2005) Role of ITAM-containing adapter proteins and their receptors in the immune system and bone. Immunol Rev 208: 50–65.M. HumphreyL. LanierM. Nakamura2005Role of ITAM-containing adapter proteins and their receptors in the immune system and bone.Immunol Rev2085065
  40. 40. Ravetch JV, Lanier LL (2000) Immune inhibitory receptors. Science 290: 84–89.JV RavetchLL Lanier2000Immune inhibitory receptors.Science2908489
  41. 41. Lanier LL (2003) Natural killer cell receptor signaling. Curr Opin Immunol 15: 308–314.LL Lanier2003Natural killer cell receptor signaling.Curr Opin Immunol15308314
  42. 42. Abram CL, Lowell CA (2007) The expanding role for ITAM-based signaling pathways in immune cells. Sci STKE 2007: re2.CL AbramCA Lowell2007The expanding role for ITAM-based signaling pathways in immune cells.Sci STKE2007re2
  43. 43. Beutler B (2004) Inferences, questions and possibilities in Toll-like receptor signalling. Nature 430: 257–263.B. Beutler2004Inferences, questions and possibilities in Toll-like receptor signalling.Nature430257263
  44. 44. Kaisho T, Akira S (2006) Toll-like receptor function and signaling. J Allergy Clin Immunol 117: 979–987.T. KaishoS. Akira2006Toll-like receptor function and signaling.J Allergy Clin Immunol117979987
  45. 45. Pasare C, Medzhitov R (2004) Toll-like receptors: Linking innate and adaptive immunity. Microbes Infect 6: 1382–1387.C. PasareR. Medzhitov2004Toll-like receptors: Linking innate and adaptive immunity.Microbes Infect613821387
  46. 46. Taylor PR, Martinez-Pomares L, Stacey M, Lin HH, Brown GD, et al. (2005) Macrophage receptors and imune recognition. Ann Rev Immunol 23: 901–944.PR TaylorL. Martinez-PomaresM. StaceyHH LinGD Brown2005Macrophage receptors and imune recognition.Ann Rev Immunol23901944
  47. 47. Robinson MJ, Sancho D, Slack EC, LeibundGut-Landmann S, Sousa CRe (2006) Myeloid C-type lectins in innate immunity. Nat Immunol 7: 1258–1265.MJ RobinsonD. SanchoEC SlackS. LeibundGut-LandmannCRe Sousa2006Myeloid C-type lectins in innate immunity.Nat Immunol712581265
  48. 48. Figdor CG, van Kooyk Y, Adema GJ (2002) C-type lectin receptors on dendritic cells and Langerhans cells. Nat Rev Immunol 2: 77–84.CG FigdorY. van KooykGJ Adema2002C-type lectin receptors on dendritic cells and Langerhans cells.Nat Rev Immunol27784
  49. 49. Fritz JH, Ferrero RL, Philpott DJ, Girardin SE (2006) Nod-like proteins in immunity, inflammation and disease. Nat Immunol 7: 1250–1257.JH FritzRL FerreroDJ PhilpottSE Girardin2006Nod-like proteins in immunity, inflammation and disease.Nat Immunol712501257
  50. 50. Mariathasan S, Monack DM (2007) Inflammasome adaptors and sensors: intracellular regulators of infection and inflammation. Nat Rev Immunol 7: 31–40.S. MariathasanDM Monack2007Inflammasome adaptors and sensors: intracellular regulators of infection and inflammation.Nat Rev Immunol73140
  51. 51. van Kooyk Y, Geijtenbeek TBH (2003) DC-SIGN: Escape mechanism for pathogens. Nat Rev Immunol 3: 697–709.Y. van KooykTBH Geijtenbeek2003DC-SIGN: Escape mechanism for pathogens.Nat Rev Immunol3697709
  52. 52. Rogers NC, Slack EC, Edwards AD, Nolte MA, Schulz O, et al. (2005) Syk-dependent cytokine induction by Dectin-1 reveals a novel pattern recognition pathway for C type lectins. Immunity 22: 507–517.NC RogersEC SlackAD EdwardsMA NolteO. Schulz2005Syk-dependent cytokine induction by Dectin-1 reveals a novel pattern recognition pathway for C type lectins.Immunity22507517
  53. 53. Gross O, Gewies A, Finger K, Schafer M, Sparwasser T, et al. (2006) Card9 controls a non-TLR signalling pathway for innate anti-fungal immunity. Nature 442: 651–656.O. GrossA. GewiesK. FingerM. SchaferT. Sparwasser2006Card9 controls a non-TLR signalling pathway for innate anti-fungal immunity.Nature442651656
  54. 54. Hara H, Ishihara C, Takeuchi A, Imanishi T, Xue L, et al. (2007) The adaptor protein CARD9 is essential for the activation of myeloid cells through ITAM-associated and Toll-like receptors. Nat Immunol 8: 619–629.H. HaraC. IshiharaA. TakeuchiT. ImanishiL. Xue2007The adaptor protein CARD9 is essential for the activation of myeloid cells through ITAM-associated and Toll-like receptors.Nat Immunol8619629
  55. 55. Schulze-Luehrmann J, Ghosh S (2006) Antigen-receptor signaling to nuclear factor κB. Immunity 25: 701–715.J. Schulze-LuehrmannS. Ghosh2006Antigen-receptor signaling to nuclear factor κB.Immunity25701715
  56. 56. Thome M (2004) CARMA1, BCL-10 and MALT1 in lymphocyte development and activation. Nat Rev Immunol 4: 348–359.M. Thome2004CARMA1, BCL-10 and MALT1 in lymphocyte development and activation.Nat Rev Immunol4348359
  57. 57. Bertin J, Guo Y, Wang L, Srinivasula SM, Jacobson MD, et al. (2000) CARD9 is a novel caspase recruitment domain-containing protein that interacts with BCL10/CLAP and activates NF-kappa B. J Biol Chem 275: 41082–41086.J. BertinY. GuoL. WangSM SrinivasulaMD Jacobson2000CARD9 is a novel caspase recruitment domain-containing protein that interacts with BCL10/CLAP and activates NF-kappa B.J Biol Chem2754108241086
  58. 58. Bernasconi NL, Onai N, Lanzavecchia A (2003) A role for Toll-like receptors in acquired immunity: Up-regulation of TLR9 by BCR triggering in naive B cells and constitutive expression in memory B cells. Blood 101: 4500–4504.NL BernasconiN. OnaiA. Lanzavecchia2003A role for Toll-like receptors in acquired immunity: Up-regulation of TLR9 by BCR triggering in naive B cells and constitutive expression in memory B cells.Blood10145004504
  59. 59. Rissoan MC, Duhen T, Bridon JM, Bendriss-Vermare N, Pâeronne C, et al. (2002) Subtractive hybridization reveals the expression of immunoglobulin-like transcript 7, Eph-B1, granzyme B, and 3 novel transcripts in human plasmacytoid dendritic cells. Blood 100: 3295–3303.MC RissoanT. DuhenJM BridonN. Bendriss-VermareC. Pâeronne2002Subtractive hybridization reveals the expression of immunoglobulin-like transcript 7, Eph-B1, granzyme B, and 3 novel transcripts in human plasmacytoid dendritic cells.Blood10032953303
  60. 60. Briáere F, Bendriss-Vermare N, Delale T, Burg S, Corbet C, et al. (2002) Origin and filiation of human plasmacytoid dendritic cells. Human immunology 63(12): 1081–1093.F. BriáereN. Bendriss-VermareT. DelaleS. BurgC. Corbet2002Origin and filiation of human plasmacytoid dendritic cells.Human immunology631210811093
  61. 61. Shigematsu H, Reizis B, Iwasaki H, Mizuno S-i, Hu D, et al. (2004) Plasmacytoid dendritic cells activate lymphoid-specific genetic programs irrespective of their cellular origin. Immunity 21: 43–53.H. ShigematsuB. ReizisH. IwasakiS-i MizunoD. Hu2004Plasmacytoid dendritic cells activate lymphoid-specific genetic programs irrespective of their cellular origin.Immunity214353
  62. 62. Schotte R, Rissoan M-C, Bendriss-Vermare N, Bridon J-M, Duhen T, et al. (2003) The transcription factor Spi-B is expressed in plasmacytoid DC precursors and inhibits T-, B-, and NK-cell development. Blood 101: 1015–1023.R. SchotteM-C RissoanN. Bendriss-VermareJ-M BridonT. Duhen2003The transcription factor Spi-B is expressed in plasmacytoid DC precursors and inhibits T-, B-, and NK-cell development.Blood10110151023
  63. 63. LeibundGut-Landmann S, Waldburger J-M, e Sousa CR, Acha-Orbea H, Reith W (2004) MHC class II expression is differentially regulated in plasmacytoid and conventional dendritic cells. Nat Immunol 5: 899–908.S. LeibundGut-LandmannJ-M WaldburgerCR e SousaH. Acha-OrbeaW. Reith2004MHC class II expression is differentially regulated in plasmacytoid and conventional dendritic cells.Nat Immunol5899908
  64. 64. Martinelli E, Cicala C, Van Ryk D, Goode DJ, Macleod K, et al. (2007) HIV-1 gp120 inhibits TLR9-mediated activation and IFN-α secretion in plasmacytoid dendritic cells. Proc Natl Acad Sci U S A 104: 3396–3401.E. MartinelliC. CicalaD. Van RykDJ GoodeK. Macleod2007HIV-1 gp120 inhibits TLR9-mediated activation and IFN-α secretion in plasmacytoid dendritic cells.Proc Natl Acad Sci U S A10433963401
  65. 65. Geijtenbeek TBH, van Vliet SJ, Koppel EA, Sanchez-Hernandez M, Vandenbroucke-Grauls CMJE, et al. (2003) Mycobacteria target DC-SIGN to suppress dendritic cell function. J Exp Med 197: 7–17.TBH GeijtenbeekSJ van VlietEA KoppelM. Sanchez-HernandezCMJE Vandenbroucke-Grauls2003Mycobacteria target DC-SIGN to suppress dendritic cell function.J Exp Med197717
  66. 66. Karp CL, Wysocka M, Wahl LM, Ahearn JM, Cuomo PJ, et al. (1996) Mechanism of suppression of cell-mediated immunity by measles virus. Science 273: 228–231.CL KarpM. WysockaLM WahlJM AhearnPJ Cuomo1996Mechanism of suppression of cell-mediated immunity by measles virus.Science273228231
  67. 67. Schroeder JT, Bieneman AP, Xiao H, Chichester KL, Vasagar K, et al. (2005) TLR9- and FceRI-mediated responses oppose one another in plasmacytoid dendritic cells by down-regulating receptor expression. J Immunol 175: 5724–5731.JT SchroederAP BienemanH. XiaoKL ChichesterK. Vasagar2005TLR9- and FceRI-mediated responses oppose one another in plasmacytoid dendritic cells by down-regulating receptor expression.J Immunol17557245731
  68. 68. Novak N, Allam J-P, Hagemann T, Jenneck C, Laffer S, et al. (2004) Characterization of FceRI-bearing CD123+ blood dendritic cell antigen-2+ plasmacytoid dendritic cells in atopic dermatitis. J Allergy Clin Immunol 114: 364–370.N. NovakJ-P AllamT. HagemannC. JenneckS. Laffer2004Characterization of FceRI-bearing CD123+ blood dendritic cell antigen-2+ plasmacytoid dendritic cells in atopic dermatitis.J Allergy Clin Immunol114364370
  69. 69. Fuchs A, Cella M, Kondo T, Colonna M (2005) Paradoxic inhibition of human natural interferon-producing cells by the activating receptor NKp44. Blood 106: 2076–2082.A. FuchsM. CellaT. KondoM. Colonna2005Paradoxic inhibition of human natural interferon-producing cells by the activating receptor NKp44.Blood10620762082
  70. 70. Blasius AL, Cella M, Maldonado J, Takai T, Colonna M (2006) Siglec-H is an IPC-specific receptor that modulates type I IFN secretion through DAP12. Blood 107: 2474–2476.AL BlasiusM. CellaJ. MaldonadoT. TakaiM. Colonna2006Siglec-H is an IPC-specific receptor that modulates type I IFN secretion through DAP12.Blood10724742476
  71. 71. Gottenberg J-E, Cagnard N, Lucchesi C, Letourneur F, Mistou S, et al. (2006) Activation of IFN pathways and plasmacytoid dendritic cell recruitment in target organs of primary Sjogren's syndrome. Proc Natl Acad Sci U S A 103: 2770–2775.J-E GottenbergN. CagnardC. LucchesiF. LetourneurS. Mistou2006Activation of IFN pathways and plasmacytoid dendritic cell recruitment in target organs of primary Sjogren's syndrome.Proc Natl Acad Sci U S A10327702775
  72. 72. Lövgren T, Eloranta M-L, Kastner B, Wahren-Herlenius M, Alm GV, et al. (2006) Induction of interferon-a by immune complexes or liposomes containing systemic lupus erythematosus autoantigen- and Sjögren's syndrome autoantigen-associated RNA. Arthritis Rheum 54: 1917–1927.T. LövgrenM-L ElorantaB. KastnerM. Wahren-HerleniusGV Alm2006Induction of interferon-a by immune complexes or liposomes containing systemic lupus erythematosus autoantigen- and Sjögren's syndrome autoantigen-associated RNA.Arthritis Rheum5419171927
  73. 73. Greenberg SA, Pinkus JL, Pinkus GS, Burleson T, Sanoudou D, et al. (2005) Interferon-alpha/beta-mediated innate immune mechanisms in dermatomyositis. Ann Neuro 57: 664–678.SA GreenbergJL PinkusGS PinkusT. BurlesonD. Sanoudou2005Interferon-alpha/beta-mediated innate immune mechanisms in dermatomyositis.Ann Neuro57664678
  74. 74. Wenzel J, Schmidt R, Proelss J, Zahn S, Bieber T, et al. (2006) Type I interferon-associated skin recruitment of CXCR3+ lymphocytes in dermatomyositis. Clin Exp Dermatol 31: 576–582.J. WenzelR. SchmidtJ. ProelssS. ZahnT. Bieber2006Type I interferon-associated skin recruitment of CXCR3+ lymphocytes in dermatomyositis.Clin Exp Dermatol31576582
  75. 75. Nestle FO, Conrad C, Tun-Kyi A, Homey B, Gombert M, et al. (2005) Plasmacytoid predendritic cells initiate psoriasis through interferon-a production. J Exp Med 202: 135–143.FO NestleC. ConradA. Tun-KyiB. HomeyM. Gombert2005Plasmacytoid predendritic cells initiate psoriasis through interferon-a production.J Exp Med202135143
  76. 76. Gilliet M, Conrad C, Geiges M, Cozzio A, Thurlimann W, et al. (2004) Psoriasis triggered by toll-like receptor 7 agonist imiquimod in the presence of dermal plasmacytoid dendritic cell precursors. Arch Dermatol 140: 1490–1495.M. GillietC. ConradM. GeigesA. CozzioW. Thurlimann2004Psoriasis triggered by toll-like receptor 7 agonist imiquimod in the presence of dermal plasmacytoid dendritic cell precursors.Arch Dermatol14014901495
  77. 77. Qin XF, An DS, Chen IS, Baltimore D (2003) Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl Acad Sci U S A 100: 183–188.XF QinDS AnIS ChenD. Baltimore2003Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5.Proc Natl Acad Sci U S A100183188