Discovery and characterization of the ligands of NK-cell receptors implicated in human diseases

Abstract

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system that act as first-line defenders against intracellular pathogens. Their functions are dictated by germline-encoded activating and inhibitory receptors, of which the most diverse family is the killer-cell immunoglobulin-like receptors (KIRs). Whereas many KIRs bind well-described HLA class I (HLA-I) ligands, many remain “orphaned” despite robust disease associations. KIR3DS1, in particular, is an activating receptor that has been associated to delayed HIV-1 disease progression, as well as the outcome of several other human diseases. However, despite knowing the ligands of its highly homologous inhibitory counterpart KIR3DL1, a ligand that accounts for the biological effects of KIR3DS1 remained unknown.

To identify HLA-I ligands of KIR3DS1, we screened 100 HLA-I proteins and found that KIR3DS1 binds HLA-F, which was validated biochemically and functionally. Primary human KIR3DS1+ NK cells exhibited a polyfunctional response upon encountering HLA-F, and suppressed HIV-1 replication in vitro. Next, we probed the cellular contexts for HLA-F expression, and found that CD4+ T-cell activation induced HLA-F expression and binding of soluble KIR3DS1-Fc. Although HLA-F was expressed intracellularly in transduced cell lines, HLA-F mobilization to the cell surface could be achieved by cellular activation and inflammatory cytokines, suggesting that HLA-F is expressed in the context of inflammation.

To ascertain non–HLA-I ligands of KIR3DS1, we found that cell lines of various tissue origins bound KIR3DS1-Fc irrespective of HLA-F expression. Using a genome-wide CRISPR/Cas9 knock-out screen, we discovered that KIR3DS1 bound to heparan sulfate proteoglycans (HSPGs), which was validated biochemically and using cell lines and primary cells. In addition, given the previously assumed binding of KIR3DS1 to HLA-B*57:01, as well as the well-document binding of KIR3DL1 to HLA-B*57:01, we investigated these interactions and found that binding of KIR3DL1 to HLA-B*57:01 is N-glycan dependent, which has not been previously described and may play a modulatory role in KIR:HLA-I interactions.

Thus, we established HLA-F and HSPGs as ligands of KIR3DS1, demonstrated cell-context–dependent expression of HLA-F and HSPGs, and revealed dependency of the HLA-I N-glycan in KIR:HLA-I interactions that may explain the widespread influence of KIR3DS1 and other NK-cell receptors in human diseases.