Siglec-H is an IPC-specific receptor that modulates type I IFN secretion through DAP12

Abstract

Natural interferon (IFN)-producing cells are the primary cell type responsible for production of type I IFN in response to viruses. Herein we report the identification of the first molecular marker of mouse natural interferon-producing cells (IPCs), a novel member of the sialic acid-binding immunoglobulin (Ig)-like lectin (Siglec) family termed Siglec-H. Siglec-H is expressed exclusively on IPCs and is unique among Siglec proteins in that it associates with the adaptor protein DAP12. Moreover, we show that DAP12 modulates the type I IFN response of IPCs to a Toll-like receptor 9 (TLR9) agonist. This observation explains our previous finding that stimulation of IPCs with 440c, a Siglec-H-specific antibody, reduces IPC secretion of type I IFN. Moreover, it supports a model in which engagement of DNAX-activation protein 12 (DAP12)-associated receptors with antibodies or low avidity endogenous ligands interferes with TLR-mediated cellular activation.

Figure 1.

Antibody 440c recognizes Siglec-H. (A) Total splenocytes from WT (C57BL/6), DAP12^-/-, DAP10^-/-, or FcRγ^-/- mice (all on C57BL/6 background) were stained with phycoerythrin (PE)-labeled mAb anti-B220 and biotinylated mAbs 440c or mPDCA1, followed by streptavidin-APC and analyzed by 2-color flow cytometry. Percentages of 440⁺ and mPDCA1⁺ IPCs are indicated. IPCs appear to be less abundant in WT mice than in DAP12^-/- mice. It is possible that the lack of 440c Ag expression in the DAP12^-/- mice influences IPC homing to the spleen, IPC development, or IPC survival. (B) 293T cells expressing murine DAP12 (293T-DAP12) and 293T cells were transiently transfected with a Siglec-H-encoding cDNA, stained with mAb 440c, and analyzed by flow cytometry. FSC, forward scatter. (C) Alignment of V-type Ig domains of Siglec-H and mouse CD33. Identical residues are darkly shaded, similar residues are lightly shaded. Residues likely involved in sialic acid binding are boxed. (D) Alignment of transmembrane regions of mouse Siglec-H, mouse CD33, and chimpanzee Siglec-13. The conserved lysine residue that may allow for DAP12 association is indicated with an asterisk.

Figure 2.

DAP12-deficient IPC secrete more IFN-α than WT IPC in vitro and in vivo. (A) Splenic CD11c⁺B220⁺CD11b^-Ly6C⁺ IPCs from WT C57BL/6 and DAP12^-/- mice were sorted by flow cytometry. Cells were stimulated with CpG oligonucleotide 2216 for 16 hours. Data are compiled from 6 independent experiments. (B) Mice received intravenous injections of 5 μg CpG 2216 in DOTAP and serum was collected after 6 hours. Representative data of 1 of 3 independent experiments are shown, each with 3 mice per group. IFN-α was measured in cell culture supernatants (A) or mouse serum by ELISA (B). Error bars show one standard deviation. ^*P < .05 versus B6 by Student t test.