Functional and Phenotypic Characterization of Siglec-6 on Human Mast Cells

Abstract

Mast cells are tissue-resident cells that contribute to allergic diseases, among others, due to excessive or inappropriate cellular activation and degranulation. Therapeutic approaches to modulate mast cell activation are urgently needed. Siglec-6 is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptor selectively expressed by mast cells, making it a promising target for therapeutic intervention. However, the effects of its engagement on mast cells are poorly defined. Siglec-6 expression and endocytosis on primary human mast cells and mast cell lines were assessed by flow cytometry. SIGLEC6 mRNA expression was examined by single-cell RNAseq in esophageal tissue biopsy samples. The ability of Siglec-6 engagement or co-engagement to prevent primary mast cell activation was determined based on assessments of mediator and cytokine secretion and degranulation markers. Siglec-6 was highly expressed by all mast cells examined, and the SIGLEC6 transcript was restricted to mast cells in esophageal biopsy samples. Siglec-6 endocytosis occurred with delayed kinetics relative to the related receptor Siglec-8. Co-crosslinking of Siglec-6 with FcεRIα enhanced the inhibition of mast cell activation and diminished downstream ERK1/2 and p38 phosphorylation. The selective, stable expression and potent inhibitory capacity of Siglec-6 on human mast cells are favorable for its use as a therapeutic target in mast cell-driven diseases.

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

“…Our findings corroborate previous studies showing Siglec-6 is highly and selectively expressed on tissue MCs and to a much lower extent, memory B cells in non-malignant samples 21,27,28,34 . Despite detectable levels of Siglec-6 on B cells, Siglec-6 mAb treatment did not induce ADCP of memory B cells.…”

“…Siglec-6 mAbs display different binding characteristics. Siglec-6 has been reported to be expressed on skin and esophageal tissue MCs, specific populations of trophoblasts, and memory B cells 21,27,28 . To confirm Siglec-6 expression, we profiled Siglec-6 surface expression on major immune cell populations in human peripheral blood as well as lung, skin, and gastrointestinal (GI) tissues by flow cytometry using a commercially available mAb (Supplemental Fig.…”

“…Interestingly, MCs express several inhibitory Siglecs, including Siglec-2 (CD22), Siglec-3 (CD33), Siglec-6, Siglec-7, and Siglec-8 17 . Previous studies have shown that engagement of CD33, Siglec-6, Siglec-7, and Siglec-8 with FcεRI induce inhibition of MC activation in vitro [17][18][19][20][21] . In addition, ligation of CD33 with ligand coated liposomes or Siglec-8 with a monoclonal antibody (mAb) reduces inflammation in vivo 22,23 .…”

“…Siglec-6 is an inhibitory receptor that is selectively expressed on human MCs and represents an attractive therapeutic target. Engagement of Siglec-6 with a mAb was recently shown to broadly inhibit MC activation in vitro 21 , suggesting binding Siglec-6 with an agonistic antibody leads to downstream inhibition. While it is well recognized that engaging Siglecs with agonistic antibodies or ligands mediates inhibition across immune cells, the mechanisms that govern this agonism are not well understood.…”

Mast cell silencing: A novel therapeutic approach for urticaria and other mast cell‐mediated diseases

“…120 Anti-Siglec-6 mAbs appear to mediate broad inhibition of IgE-dependent and -independent MC activation and degranulation through multiple routes, including FcεRI, complement component 5a receptor, and MRGPRX2 pathways. 121 Consistent with the inhibitory function of Siglec-6, mutation of both the ITIM and ITIM-like domains abrogate anti-Siglec-6-mediated MC inhibition by preventing SHP-1 and SHP-2 phosphatase recruitment. 122 In humanized mice, anti-Siglec-6 mAbs reduced degranulation and soluble mediator production of activated human MCs in vitro and inhibited IgE-mediated systemic anaphylaxis.…”