BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates antigen capture and is a potent inhibitor of interferon alpha/beta induction

Abstract

Plasmacytoid dendritic cells are present in lymphoid and nonlymphoid tissue and contribute substantially to both innate and adaptive immunity. Recently, we have described several monoclonal antibodies that recognize a plasmacytoid dendritic cell-specific antigen, which we have termed BDCA-2. Molecular cloning of BDCA-2 revealed that BDCA-2 is a novel type II C-type lectin, which shows 50.7% sequence identity at the amino acid level to its putative murine ortholog, the murine dendritic cell-associated C-type lectin 2. Anti-BDCA-2 monoclonal antibodies are rapidly internalized and efficiently presented to T cells, indicating that BDCA-2 could play a role in ligand internalization and presentation. Furthermore, ligation of BDCA-2 potently suppresses induction of interferon alpha/beta production in plasmacytoid dendritic cells, presumably by a mechanism dependent on calcium mobilization and protein-tyrosine phosphorylation by src-family protein-tyrosine kinases. Inasmuch as production of interferon alpha/beta by plasmacytoid dendritic cells is considered to be a major pathophysiological factor in systemic lupus erythematosus, triggering of BDCA-2 should be evaluated as therapeutic strategy for blocking production of interferon alpha/beta in systemic lupus erythematosus patients.

Figure 4.

(A) Identification of putative alternative splice forms of BDCA-2 mRNA. Poly(A)⁺ RNA was isolated from purified PDCs and analyzed for the presence of BDCA-2 mRNA by RT-PCR amplification (lane 2, 20 PCR cycles; lane 3, 25 PCR cycles; lane 4, 30 PCR cycles). The PCR products were size-fractionated by 4–12% TBE PAGE. A further PCR amplification of individual excised bands enabled the cloning and sequencing of individual splice variants. (B) Schematic drawing of the coding region of full-length BDCA-2 mRNA. The structural domains are indicated by different colors (violet, cytoplasmic domain; red, transmembrane domain; green, neck domain; and blue, CRD) and the individual exons (exons 2–7) of the coding region are represented as boxes. Below the full-length BDCA-2 mRNA, BDCA-2 splice variants are shown with the missing exons indicated by gaps.

Figure 1.

Amino acid sequence alignment of the type II C-type lectins BDCA-2, murine dectin-2, and human DCIR. Identical or conserved residues are indicated by (*), conserved and semiconserved substitutions by (:) and (.), respectively; the putative transmembrane domains are shown in red italics; the shaded area denotes the CRD and the residues strongly conserved among C-type lectins are shown in bold type (H, hydrophobic; A, aliphatic; C, cysteine; G, glycine; E, glutamic acid; W, tryptophan; Δ, aromatic; +, involved in calcium-dependent binding of carbohydrate; +P++, so-called EPN-motif predicting mannose-type specificity [reference 46]).

Figure 2.

Expression analysis of BDCA-2 mRNA by RT-PCR (34 PCR-cycles) on various tissues and cell populations (1, heart; 2, brain; 3, placenta; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, pancreas; 9, spleen; 10, thymus; 11, testis; 12, ovary; 13, small intestine; 14, LN; 15, bone marrow; 16, fetal liver; 17, tonsil; 18, T cells; 19, B cells; 20, NK cells; 21, monocytes; 22, CD11c^brightCD123^low myeloid DCs (reference 19); 23, CD11c⁻CD123^bright PDCs). All cDNAs were normalized using four housekeeping genes including glyceraldehyde 3-phosphate dehydrogenase (G3PDH).

Figure 3.

Anatomical localization of BDCA-2–expressing PDCs in inflamed tonsils. Fluorescent double staining with FITC–conjugated (green) anti–BDCA-2 mAb and Texas Red–conjugated (red) CD8 mAb (A), CD20 mAb (B), CD123 mAb (C), and anti–HLA-DR mAb (D). Note that BDCA-2–expressing PDCs are found in the T cell-rich extrafollicular areas but not within the germinal center. Like CD123, BDCA-2 is expressed on PDCs, but unlike CD123, BDCA-2 is not expressed on HEVs. One representative experiment of three is shown.

Figure 5.

SDS-PAGE analysis of BDCA-2 immunoprecipitated from ¹²⁵I-labeled PDC (A) and BDCA-2–transfected U937 human monocytoid leukemia cells (B). BDCA-2 appears as a ∼38 kD band under nonreducing (NR) and reducing (R) conditions. IC, isotype-matched control mAb. One representative experiment of three is shown.

Figure 6.

A rapid and transient rise in [Ca²⁺]i is induced in PDCs (left dotplots) and BDCA-2-transfected U937 cells (middle dotplots), but not in nontransfected U937 cells (right dotplots) after ligation of surface BDCA-2 with specific primary mAb (AC144, IgG1) and secondary cross-linking F(ab′)₂ goat anti–mouse IgG (B). This [Ca²⁺]i increase is not affected when extracellular calcium is chelated with excess EGTA (C), but inhibited when src-family protein-tyrosine kinases are blocked by preincubation with the specific inhibitor PP2 (D). One representative experiment of six is shown.

Figure 7.

Triggering of BDCA-2 induces protein tyrosine phosphorylation in purified PDCs (A) and BDCA-2 transfected U937 cells (B), but not in BDCA-2–transfected Jurkat cells (C). Cells were incubated with medium alone (−) or with anti–BDCA-2 mAb (AC144, IgG1) (+). Cell lysates were size-fractionated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with horseradish peroxidase-coupled antiphosphotyrosine mAb PY20. One representative experiment of two is shown.

Figure 8.

Presentation of anti–BDCA-2 mAb (AC144, IgG1) to a T cell clone specific for mouse IgG1 by irradiated PDCs. Anti–BDCA-2 mAb AC144 (▪) is presented more efficiently than anti-ILT3 mAb ZM3.8 (▴) and far more efficiently than anti-cytokeratin mAb CK3–11D5 (•). One representative experiment of two is shown.

Figure 9.

(A) Ligation of BDCA-2 suppresses induction of IFN-α/β production in PDCs. Stimulation of PDCs with FLU, anti-ss/ds DNA mAb MER-3 plus plasmid pcDNA3, or serum from a SLE patient, but not with anti-ss/ds DNA mAb MER-3 alone or plasmid pcDNA3 alone, induces production of large amounts of IFN-α/β in PDCs. Induction of IFN-α/β production with this agents can be inhibited by coincubation with anti–BDCA-2 mAb (AC144, IgG1), but not by coincubation with an isotype-matched control IgG1 mAb. The data shown are representative of more than six experiments using FLU, more than two experiments using anti-ss/ds DNA mAb MER-3 plus plasmid pcDNA3, and three experiments using SLE sera as IFN-α/β production–inducing agent. (B) Anti–BDCA-2 mAb-mediated suppression of induction of IFN-α/β production is mAb concentration dependent. Purified PDCs were coincubated with one of the IFN-α/β–inducing agents (anti-ss/ds DNA mAb MER-3 plus plasmid pcDNA3) and titrated amounts of anti–BDCA-2 mAb (AC144, IgG1). Note that concentrations below 100 ng/ml are sufficient for a 50% inhibition of the IFN-α/β response.