Genome-wide CRISPR screens reveal a specific ligand for the glycan-binding immune checkpoint receptor Siglec-7

Abstract

Significance Cancer cells are frequently coated with chains of sugar molecules (glycans) that bind to inhibitory immune receptors, leading to suppression of anticancer immunity. While blocking these interactions may be therapeutically beneficial, the specific glycoprotein ligands that engage such receptors are often complex and difficult to characterize. To solve this problem, we used unbiased genome-wide screening to identify genes required for cell-surface presentation of ligands that bind members of the Siglec immune receptor family. This approach led to identification of the glycoprotein CD43 as a highly specific ligand for Siglec-7. Blocking the interaction between CD43 and Siglec-7 sensitizes leukemia cells to immune cell lysis, implying that targeting the CD43/Siglec-7 checkpoint could be therapeutically beneficial. Glyco-immune checkpoint receptors, molecules that inhibit immune cell activity following binding to glycosylated cell-surface antigens, are emerging as attractive targets for cancer immunotherapy. Defining biologically relevant ligands that bind and activate such receptors, however, has historically been a significant challenge. Here, we present a CRISPRi genomic screening strategy that allowed unbiased identification of the key genes required for cell-surface presentation of glycan ligands on leukemia cells that bind the glyco-immune checkpoint receptors Siglec-7 and Siglec-9. This approach revealed a selective interaction between Siglec-7 and the mucin-type glycoprotein CD43. Further work identified a specific N-terminal glycopeptide region of CD43 containing clusters of disialylated O-glycan tetrasaccharides that form specific Siglec-7 binding motifs. Knockout or blockade of CD43 in leukemia cells relieves Siglec-7-mediated inhibition of immune killing activity. This work identifies a potential target for immune checkpoint blockade therapy and represents a generalizable approach to dissection of glycan–receptor interactions in living cells.