PROJECT SUMMARY/ABSTRACT Regulation of the immune system is substantially influenced by glycosylation. Cell-surface glycans tune ligand-receptor binding and set a threshold for initiating the downstream signaling for immune cell activation. Siglecs (sialic acid-binding immunoglobulin-type lectins) are a family of regulatory receptors involved in these processes. A Siglec can bind to both cis and trans sialylated glycan ligands that are expressed on the same or interacting cells, respectively. The binding of Siglecs with their ligands can either segregate Siglecs from activation receptors or move them closer. The ability of inhibitory Siglecs to modulate activation receptors is regulated by spatial proximity: recruiting Siglecs to the immune synapse in the proximity of activation receptors would trigger inhibitory signaling to suppress immune-system activation, whereas moving Siglecs away from activation receptors would enable optimal signaling through activation receptors. For the above reasons, recently, Siglecs have been described as glyco-immune checkpoints. Through their interaction with sialylated glycans aberrantly expressed on tumor cells, innate immune cell-associated Siglecs trigger signaling cascades to inhibit immune-system activation. Likewise, Siglecs upregulated on tumor cells interact with yet-to-be identified T-cell membrane glycoproteins to suppress T cell anti-tumor functions. On the positive side, however, inhibitory signaling through Siglecs curbs inflammation during cell death induced by viral infection. Despite these intriguing observations, the mechanisms underlying the above processes are just starting to be elucidated. The overarching goal of this project is to use a combination of chemoenzymatic, biochemical and genetic tools to explore Siglec-glycan ligand interactions and their therapeutic implication. In Aim 1, we will design Siglec-based chimeric switch receptors and convert inhibitory Siglecs into activation receptors. In Aim 2, we will use a cell-based glycan array platform to screen for high-affinity and specific ligands of Siglecs. Once identified, we will explore their utilities to suppress or harness the inhibitory Siglec signaling for therapeutic applications. Finally, we will use our chemoenzymatic tools to investigate how the Siglec-cis ligand interaction is involved in mediating the Siglec–trans ligand interaction and accordingly immune cell activation (Aim 3).