Sialic acid cis-ligand dynamics modulate Siglec-7 and -9 function and affect Siglec-7/9 co-blockade to potentiate natural killer cell anti-tumor activity.

The glyco-immune checkpoints Siglec-7 and Siglec-9 have received considerable interest as targets for cancer immunotherapy. How Siglec-7/9-sialic acid cis-interactions on immune cells influence trans-signaling induced by tumor cells and whether Siglec-7 and -9 co-blockade can enhance immune effector cell function are key questions for clinical translation. We developed and applied single and dual Jurkat/MA NFAT-luciferase reporter cells expressing wild-type or mutant chimeric Siglec-7 and/or -9. Cis-interactions on these Jurkat/MA reporter cells prevented Siglec-7 and -9 signaling induced by trans-ligands, i.e. on tumor cells. In Jurkat/MA cells expressing both receptors, Siglec-7/9 co-inhibition was essential to fully block receptor signaling. Extrapolating our findings to human primary cells, NK cell-mediated killing of melanoma and acute myeloid leukemia (AML) cell lines and patient-derived AML cells was increased upon Siglec-7 and/or -9 blockade. Importantly, co-blockade was superior to single blocking strategies and the effects were most pronounced when cis-ligands were removed from the NK cell' surface using sialidase. Further diving into cis-ligand dynamics on primary human NK cells, physiological NK cell activation with IL-2 or IFN-α or IL-15/IL-2-induced proliferation was shown to significantly downregulate Siglec-7 and -9 cis-ligand expression. Moreover, Siglec-7 and -9 ligands were progressively downregulated with each round of NK cell division. Taken together, our findings highlight the important role of cis-interactions in regulating trans-interactions and emphasize the potential of simultaneously blocking Siglec-7 and -9 for clinical applications. These insights may guide the design of next-generation Siglec-targeted immunotherapies.