Dissecting the Ability of Siglecs To Antagonize Fc Receptors

Fc gamma receptors (Fc gamma Rs) play key roles in the effector function of IgG, but their inappropriate activation plays a role in several disease etiologies. Therefore, it is critical to better understand how Fc gamma Rs are regulated. Numerous studies suggest that sialic acid-binding immunoglobulin-type lectins (Siglecs), a family of immunomodulatory receptors, modulate Fc gamma R activity; however, it is unclear of the circumstances in which Siglecs can antagonize Fc gamma Rs and which Siglecs have this ability. Using liposomes displaying selective ligands to coengage Fc gamma Rs with a specific Siglec, we explore the ability of Siglec-3, Siglec-5, Siglec-7, and Siglec-9 to antagonize signaling downstream of Fc gamma Rs. We demonstrate that Siglec-3 and Siglec-9 can fully inhibit Fc gamma R activation in U937 cells when coengaged with Fc gamma Rs. Cells expressing Siglec mutants reveal differential roles for the immunomodulatory tyrosine-based inhibitory motif (ITIM) and immunomodulatory tyrosine-based switch motif (ITSM) in this inhibition. Imaging flow cytometry enabled visualization of SHP-1 recruitment to Siglec-3 in an ITIM-dependent manner, while SHP-2 recruitment is more ITSM-dependent. Conversely, both cytosolic motifs of Siglec-9 contribute to SHP-1/2 recruitment. Siglec-7 poorly antagonizes Fc gamma R activation for two reasons: masking by cis ligands and differences in its ITIM and ITSM. A chimera of the Siglec-3 extracellular domains and Siglec-5 cytosolic tail strongly inhibits Fc gamma R when coengaged, providing evidence that Siglec-5 is more like Siglec-3 and Siglec-9 in its ability to antagonize Fc gamma Rs. Additionally, Siglec-3 and Siglec-9 inhibited Fc gamma Rs when coengaged by cells displaying ligands for both the Siglec and Fc gamma Rs. These results suggest a role for Siglecs in mediating Fc gamma R inhibition in the context of an immunological synapse, which has important relevance to the effectiveness of immunotherapies.