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

Abstract 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-like transcript 3/leukocyte immunoglobulin-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 was identified very recently as fibronectin, particularly the N-terminal 30 kDa domain (FN30). We hypothesized that LILRB4 could bind fibronectin on target cells in trans together with integrins, classical fibronectin receptors, 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 integrins suggested that these novel and classical fibronectin receptors, respectively, co-engage fibronectin 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 fibronectin. While surface fibronectin-poor YAC-1 cells were evenly sensitive as to natural killing of both gp49B-positive and -negative NK cells, the killing of fibronectin-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 fibronectin on target cells, which sheds light on the unexpected role of LILRB4 and fibronectin as a potential attenuator of NK cell cytotoxicity in the tumor microenvironment. Fibronectin-LILRB4 interactions attenuate NK cell cytotoxicity Graphical Abstract Fibronectin (FN) on target cells attenuates NK cytotoxicity via LILRB4. People understand that fibronectin in the extracellular matrix of tumors endows the tumor cells a sticky nature, which is advantageous for their metastasis and growth. In this study, we found that FN+ tumor cells escape from potential attack by NK cells. Thus, natural cytotoxicity of NK cells is tightly regulated by FN on target cells. The key inhibitory receptor on NK cells is LILRB4/gp49B, a myeloid immune checkpoint. LILRB4/gp49B plays such an important role with their partner activating-type receptors, integrins. These novel findings reveal the unexpected mechanism for attenuat