Regulation of mast cells by overlapping but distinct protein interactions of Siglec-6 and Siglec-8.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec)-6 and Siglec-8 are closely related mast cell (MC) receptors with broad inhibitory activity, but whose functional differences are incompletely understood. METHODS: Proteomic profiling using quantitative mass spectrometry was performed on primary mouse MCs to identify proteins associated with Siglec-6 and Siglec-8. For functional characterization, each receptor was evaluated biochemically and in ex vivo and in vivo inhibition models of IgE and non-IgE-mediated MC activation in Siglec-6- or Siglec-8-expressing transgenic mice. RESULTS: Siglec-6 and Siglec-8 were found in MCs within large complexes, interacting with 66 and 86 proteins, respectively. Strikingly, Siglec-6 and Siglec-8 interacted with a large cluster of proteins involved in IgE and non-IgE-mediated MC activation, including the high affinity IgE receptor, stem cell factor (SCF) receptor KIT/CD117, IL-4 and IL-33 receptors, and intracellular kinases LYN and JAK1. Protein interaction networks revealed Siglec-6 and Siglec-8 had overlapping yet distinct MC functions, with a potentially broader regulatory role for Siglec-6. Indeed, Siglec-6 preferentially interacted with the mature form of KIT at the cell surface, and treatment with an anti-Siglec-6 antibody significantly inhibited SCF-mediated MC activation more in comparison to targeting Siglec-8. CONCLUSION: These data demonstrate a central role for Siglec-6 and Siglec-8 in controlling MC activation through interactions with multiple activating receptors and key signaling molecules. Our findings suggest that Siglec-6 has a role distinct from that of Siglec-8 in regulating MC function and represents a distinct potential therapeutic target in mast cell-driven diseases.

Discovery of an agonistic Siglec-6 antibody that inhibits and reduces human mast cells.

Mast cells (MC) are key drivers of allergic and inflammatory diseases. Sialic acid-binding immunoglobulin-like lectin (Siglec)-6 is an immunoregulatory receptor found on MCs. While it is recognized that engaging Siglecs with antibodies mediates inhibition across immune cells, the mechanisms that govern this agonism are not understood. Here we generated Siglec-6 mAb clones (AK01 to AK18) to better understand Siglec-6-mediated agonism. Siglec-6 mAbs displayed epitope-dependent receptor internalization and inhibitory activity. We identified a Siglec-6 mAb (AK04) that required Fc-mediated interaction for receptor internalization and induced inhibition and antibody-dependent cellular phagocytosis against MCs. AK04-mediated MC inhibition required Siglec-6 immunoreceptor tyrosine-based inhibitory motif (ITIM) and ITIM-like domains and was associated with receptor cluster formation containing inhibitory phosphatases. Treatment of humanized mice with AK04 inhibited systemic anaphylaxis with a single dose and reduced MCs with chronic dosing. Our findings suggest Siglec-6 activity is epitope dependent and highlight an agonistic Siglec-6 mAb as a potential therapeutic approach in allergic disease.