Inhibition of Natural Killer Cell Function

National Institute Of Allergy And Infectious Diseases

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Abstract

Human natural killer (NK) cell activity is regulated by a family of killer-cell Ig-like receptors (KIR) for HLA class I. Combinations of KIR and HLA genotypes are associated with disease susceptibility, including sensitivity to virus infection and disorders of pregnancy. The goal of our study was to determine the extent to which peptides bound to HLA-C influence the specific binding of inhibitory KIR to group C1 or C2 HLA-C allotypes. Endogenous HLA-C*05:01-associated peptides were identified and tested for KIR binding to peptide-loaded HLA-C*05:01 (C2) and HLA-C*08:02 (C1), which are canonical ligands for KIR2DL1 and KIR2DL3, respectively, despite having only two amino acid differences. Specific binding of KIR2DL1 to the C2 allotype occurred with most peptides tested. In contrast, KIR2DL3 binding to the C1 allotype was restricted to a subset of peptides, providing an explanation for why KIR2DL3C1 interactions appear to be weaker than KIR2DL1C2. Furthermore, cross-reactive and functional binding of KIR2DL3 with the C2 allotype was even more restricted, occurring with only a few peptides. Moreover, we identified two peptides that, unexpectedly, promoted binding of the C2 allotype-specific KIR2DL1 to the C1 allotype. Our data show that KIR binding to HLA-C in the context of multiple peptides is a feature of strong specificity for HLA-C allotype C2, whereas greater selectivity for peptides is associated with more permissive reactivity with HLA-C allotypes. Peptide sequence-driven binding of KIR could lead to disease associations of KIR genotypes with specific HLA alleles, and be exploited by pathogens to modulate NK cell responses. The inhibitory function of killer cell immunoglobulin-like receptors (KIR) that bind HLA-C and block activation of human natural killer (NK) cells is dependent on zinc. We have reported that zinc induced the assembly of soluble KIR into filamentous polymers, as detected by electron microscopy, which depolymerized after zinc chelation. Similar KIR filaments were isolated from lysates of cells treated with zinc, and membrane protrusions enriched in zinc were detected on whole cells by scanning electron microscopy and imaging mass spectrometry. Two independent mutations in the extracellular domain of KIR, away from the HLA-C binding site, impaired zinc-driven polymerization and inhibitory function. KIR filaments formed spontaneously, without addition of zinc, at functional inhibitory immunological synapses of NK cells with HLA-C+ cells. As zinc is toxic, its availability is tightly regulated by transporters and zinc-binding proteins. Most of the zinc in the serum is sequestered by zinc-binding proteins. To test how zinc is delivered to KIR at inhibitory immunological synapses, live imaging was performed to visualize 'free' (i.e. bioavailable) zinc in NK cells and to follow the localization and movement of zinc transporters. The transporter ZnT1 is the main zinc channel at the plasma membrane for extrusion of zinc out of cells. A ZnT1 tagged with a fluorescent protein was transfected into an NK cell line that expresses an HLA-C-specific inhibitory KIR. At activating immunological synapses between NK and target cells ZnT1 remained evenly distributed at the plasma membrane. Strikingly, ZnT1 polarized and accumulated at inhibitory immunological synapses with HLA-C+ target cells, suggesting that it provides the zinc required for inhibitory KIR function.