BDCA2/Fc epsilon RI gamma complex signals through a novel BCR-like pathway in human plasmacytoid dendritic cells

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 Fc epsilon RI gamma. 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 Fc epsilon RI gamma, BDCA2 activates a novel BCR-like signaling pathway to regulate the immune functions of pDCs.

Figure 1. BDCA2 Associates with FcɛRIγ 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, FcɛRIγ, DAP12, DAP10, or a Y-F ITAM mutant FcɛRIγ. The surface staining by isotype-matched control mAb is shown as a shaded area. (C) Co-immunoprecipitation of BDCA2 and FcɛRIγ from BDCA2 and FcɛRIγ doubly transfected Jurkat cells after cell surface biotinylation. Western blot analysis was performed with anti-FcɛRIγ Ab or with HRP-conjugated NeutrAvidin. (D) Co-immunoprecipitation of BDCA2 and FcɛRIγ from freshly isolated pDCs. Western blot analysis was performed with anti-FcɛRIγ Ab.

Figure 2. Crosslinking the BDCA2/FcɛRIγ 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) FcɛRIγ. (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.

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.

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.

Figure 5. BDCA2/FcɛRIγ 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.

Figure 6. BDCA2/FcɛRIγ 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.

Figure 7. Ligation of the BDCA2/FcɛRIγ 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.