Fibronectin on target cells attenuates natural cytotoxicity of NK cells via myeloid immune checkpoint ILT3/LILRB4/gp49B

Natural killer (NK) cells play pivotal roles in innate immunity as well as in anti-tumor responses via natural killing, while their activity is tightly regulated by cell-surface inhibitory receptors. Immunoglobulin (Ig)-like transcript 3/leukocyte Ig-like receptor B4 (ILT3/LILRB4, also known as gp49B in mice) is an inhibitory receptor expressed on activated NK cells as well as myeloid-lineage cells. The common physiologic ligand of human LILRB4 and gp49B is identified very recently as fibronectin (FN), particularly the N-terminal 30 kDa domain (FN30). We hypothesized that LILRB4 could bind FN on target cells in trans together with integrin, a classical FN receptor, in cis and deliver an inhibitory signal in NK cells, leading to attenuated natural killing. Flow cytometric and confocal microscopic analyses of NK cell-surface gp49B and integrin suggested that these novel and classical FN receptors, respectively, co-engage FN immobilized on a culture plate. Biochemical analyses indicated that tyrosine phosphorylation of spleen tyrosine kinase was augmented in gp49B-deficient NK cells upon binding to the immobilized FN. While surface FN-poor YAC-1 cells were evenly sensitive as to natural killing of both gp49B-positive and -negative NK cells, the killing of FN-rich Lewis lung carcinoma cells, but not the FN30-knockout cells, was augmented among gp49B-deficient NK cells. These results suggest that the natural cytotoxicity of NK cells is negatively regulated through LILRB4/gp49B sensing FN on target cells, which sheds light on the unexpected role of LILRB4 and FN as a potential attenuator of NK cell cytotoxicity in the tumor microenvironment.